Polynucleotides encoding citrin for the treatment of citrullinemia type 2

ABSTRACT

The invention relates to mRNA therapy for the treatment of Citrullinemia Type 2 (“CTLN2”). mRNAs for use in the invention, when administered in vivo, encode human Citrin, isoforms thereof, functional fragments thereof, and fusion proteins comprising Citrin. mRNAs of the invention are preferably encapsulated in lipid nanoparticles (LNPs) to effect efficient delivery to cells and/or tissues in subjects, when administered thereto. mRNA therapies of the invention increase and/or restore deficient levels of Citrin expression and/or activity in subjects. mRNA therapies of the invention further decrease levels of biomarkers associated with deficient Citrin activity in subjects, namely ammonia and/or triglycerides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/302,360, filed on Nov. 16, 2018, issued as U.S. Pat. No. 10,993,918,which is the National Stage of International Application No.PCT/US2017/033421, filed on May 18, 2017, which claims the prioritybenefit of U.S. Provisional Application No. 62/338,479, filed on May 18,2016. The disclosures of the prior applications are incorporated hereinby reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3529_072PC02_Sequence Listing, Size: 181,746 bytes; and Date ofCreation: May 16, 2017) is herein incorporated by reference in itsentirety.

BACKGROUND

Citrullinemia is an autosomal recessive urea cycle disorder that causesabnormal buildup of ammonia and other toxic substances in the blood.Citrullinemia Type 2 (“CTLN2”) is a form of Citrullinemia that is causedby mutation of the Solute Carrier Family 25, Member 13 gene(“SLC25A13”). CTLN2 is also known as Adult-Onset Citrullinemia Type 2 orCitrin Deficiency. CTLN2 causes nocturnal delirium, aggression,irritability, hyperactivity, delusions, disorientation, restlessness,drowsiness, loss of memory, flapping tremor, convulsive seizures, coma,or even death resulting from brain edema. Kobayashi, K. et al.,GeneReviews (2008); http://www.ncbi.nlm.nih.gov/books/NBK1181/. CTLN2 isfound in the Japanese population with an estimated incidence of 1 in100,000 to 230,000. Additionally, CTLN2 has been reported in East Asianand Middle Eastern populations. Current treatments for CTLN2 attempt tocontrol signs and symptoms by having individuals consume diets that arelow in carbohydrates and high in proteins and fats. Also, treatment witharginine, sodium benzoate, or sodium phenylacetate removes ammonia fromthe blood. In serious cases, liver transplantation has been successfulin treating CTLN2 and dialysis can be used to remove ammonia from theblood. Saheki, T. et al., Molec. Genet. Metab. 100(Suppl. 1):S59-S64(2010). Thus, most effective treatment for this disorder is livertransplantation, although many patients can be managed by alow-carbohydrate/high-protein diet. However, none of these treatmentscompletely control the disorder.

The principal gene associated with CTLN2 is SLC25A13, which has twovariants (Genbank Accession Nos. NM_001160210, NP_001153682; NM_014251,NP_055066; XM_006715831, XP_006715894; XM_011515727, XP_011514029).SLC25A13 encodes Citrin, which is a calcium-dependent mitochondrialsolute carrier protein. Citrin is also known as Mitochondrial AspartateGlutamate Carrier 2 or ARALAR2. Citrin plays a critical role in the ureacycle by catalyzing the mitochondrial uptake of glutamate and export ofaspartate. Saheki, T. et al., Metab. Brain Dis. 17:335-346 (2002).Depending on the variant, the precursor form of human Citrin is either676 or 675 amino acids, while its mature form is either 675 or 674 aminoacids with the initiator methionine cleaved off.

A complete or partial loss of Citrin function leads to abnormal buildupof ammonia and other toxic substances in the blood. For example, loss ofCitrin has been reported to lead to ammonia levels greater than 750μg/dL, as compared to 49 μg/dL or less in normal individuals. Aside fromliver transplant, treatment for CTLN2 generally focuses on managing thedisease, e.g., through diet and nutritional support. There are currentlyno available therapeutics to treat CTLN2. Accordingly, there is a needfor improved therapy to treat CTLN2.

BRIEF SUMMARY

The present invention provides mRNA therapeutics for the treatment ofCitrullinemia Type 2 (CTLN2). The mRNA therapeutics of the invention areparticularly well-suited for the treatment of CTLN2 as the technologyprovides for the intracellular delivery of mRNA encoding Citrin followedby de novo synthesis of functional Citrin protein within target cells.The instant invention features the incorporation of modified nucleotideswithin therapeutic mRNAs to (1) minimize unwanted immune activation(e.g., the innate immune response associated with the in vivointroduction of foreign nucleic acids) and (2) optimize the translationefficiency of mRNA to protein. Exemplary aspects of the inventionfeature a combination of nucleotide modifications to reduce the innateimmune response and sequence optimization, in particular, within theopen reading frame (ORF) of therapeutic mRNAs encoding Citrin to enhanceprotein expression.

In further embodiments, the mRNA therapeutic technology of the instantinvention also features delivery of mRNA encoding Citrin via a lipidnanoparticle (LNP) delivery system. The instant invention features novelionizable lipid-based LNPs which have improved properties when combinedwith mRNA encoding CTLN2 and administered in vivo, for example, cellularuptake, intracellular transport and/or endosomal release or endosomalescape. The LNPs of the invention also demonstrate reducedimmunogenicity associated with the in vivo administration of LNPs.

In certain aspects, the invention relates to compositions and deliveryformulations comprising a polynucleotide, e.g., a ribonucleic acid(RNA), e.g., a messenger RNA (mRNA), encoding Citrin and methods fortreating Citrullinemia Type 2 (CTLN2) in a subject in need thereof byadministering the same.

The present disclosure provides a pharmaceutical composition comprisinga lipid nanoparticle encapsulated mRNA that comprises an open readingframe (ORF) encoding an Citrin polypeptide, wherein the composition issuitable for administration to a human subject in need of treatment forCitrullinemia Type 2 (CTLN2).

The present disclosure further provides a pharmaceutical compositioncomprising: (a) a mRNA that comprises (i) an open reading frame (ORF)encoding an Citrin polypeptide, wherein the ORF comprises at least onechemically modified nucleobase, sugar, backbone, or any combinationthereof, (ii) an untranslated region (UTR) comprising a microRNA (miRNA)binding site; and (b) a delivery agent, wherein the pharmaceuticalcomposition is suitable for administration to a human subject in need oftreatment for Citrullinemia Type 2 (CTLN2).

The present disclosure further provides a pharmaceutical compositioncomprising an mRNA comprising an open reading frame (ORF) encoding ahuman Citrin polypeptide, wherein the composition when administered to asubject in need thereof as a single intravenous dose is sufficient toreduce blood and/or plasma levels of (i) ammonia at least 2-fold, atleast 3-fold, at least 5-fold, at least 10-fold, at least 15-fold, atleast 20-fold, or at least 50-fold compared to the subject's baselinelevel or a reference ammonia blood and/or plasma level, for at least 24hours, at least 48 hours, at least 72 hours, at least 96 hours, or atleast 120 hours post-administration; and/or (ii) triglycerides at least1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 10-fold, or at least 20-fold compared to the subject'sbaseline level or a reference triglyceride blood and/or plasma level,for at least 24 hours post-administration, at least 48 hours, at least72 hours, at least 96 hours, or at least 120 hours post-administration.

The present disclosure further provides a pharmaceutical compositioncomprising an mRNA comprising an open reading frame (ORF) encoding ahuman Citrin polypeptide, wherein the composition when administered to asubject in need thereof as a single intravenous dose is sufficient toreduce blood and/or plasma levels of (i) ammonia at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95% or at least98%, at least 99%, or 100% compared to the subject's baseline level or areference ammonia blood and/or plasma level (e.g., in a subject withCTLN2), for at least 24 hours, at least 48 hours, at least 72 hours, atleast 96 hours, or at least 120 hours post-administration; and/or (ii)triglycerides at least 30%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or at least 98%, at least 99%, or100% compared to the subject's baseline level or a referencetriglyceride blood and/or plasma level (e.g., in a subject with CTLN2),for at least 24 hours post-administration, at least 48 hours, at least72 hours, at least 96 hours, or at least 120 hours post-administration.

The present disclosure further provides a pharmaceutical compositioncomprising an mRNA comprising an open reading frame (ORF) encoding ahuman Citrin polypeptide, wherein the composition when administered to asubject in need thereof as a single intravenous dose is sufficient to:(i) increase hepatic Citrin activity level at or above a normalphysiological level for at least 24 hours, at least 48 hours, at least72 hours, at least 96 hours, or at least 120 hours post-administration,and/or (ii) increase hepatic Citrin activity level to 50% or more of areference hepatic Citrin activity level for at least 24 hours, at least48 hours, at least 72 hours, or at least 96 hours post-administration.

In some embodiments, the pharmaceutical compositions disclosed hereinfurther comprise a delivery agent.

In some aspects, the invention relates to a polynucleotide comprising anopen reading frame (ORF) encoding a Citrin polypeptide, wherein theuracil or thymine content of the ORF relative to the theoretical minimumuracil or thymine content of a nucleotide sequence encoding the Citrinpolypeptide (% U_(TM) or % T_(TM)), is between about 100% and about150%. In some aspects, the uracil or thymine content of the ORF relativeto the uracil or thymine content of the corresponding wild-type ORF (%U_(WT) or % T_(WT)) is less than 100%. In some aspects, the uracil orthymine content in the ORF relative to the total nucleotide content inthe ORF (% U_(TL) or % T_(TL)) is less than about 50%, less than about40%, less than about 30%, or less than about 20%.

In some aspects, the guanine content of the ORF with respect to thetheoretical maximum guanine content of a nucleotide sequence encodingthe Citrin polypeptide (% G_(TMX)) is at least 69%, at least 70%, atleast 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or about 100%. In some aspects, the cytosine contentof the ORF relative to the theoretical maximum cytosine content of anucleotide sequence encoding the Citrin polypeptide (% C_(TMX)) is atleast 60%, at least 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or about 100%. In some aspects, the guanine and cytosine content(G/C) of the ORF relative to the theoretical maximum G/C content of anucleotide sequence encoding the Citrin polypeptide (% G/C_(TMX)) is atleast about 81%, at least about 85%, at least about 90%, at least about95%, or about 100%. In some aspects, the G/C content in the ORF relativeto the G/C content in the corresponding wild-type ORF (% G/CWT) is atleast 102%, at least 103%, at least 104%, at least 105%, at least 106%,at least 107%, at least 110%, at least 115%, at least 120%, at least125%, at least 130%, at least 131%, at least 132%, at least 133%, or atleast 134%. In some aspects, the average G/C content in the 3^(rd) codonposition in the ORF is at least 30%, at least 35%, at least 40%, atleast 41%, at least 42%, at least 43%, at least 44%, at least 45%, atleast 46%, at least 47%, at least 48%, or at least 49% higher than theaverage G/C content in the 3^(rd) codon position in the correspondingwild-type ORF.

In some aspects, the ORF has at least 74%, at least 75%, at least 76%,at least 77%, at least 78%, at least 79%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NOs: 5-29, 129, 132, 135, or 138.

In some aspects, the Citrin polypeptide comprises an amino acid sequenceat least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or about 100% identical to (i) thepolypeptide sequence of wild type Citrin, isoform 1 (SEQ ID NO: 1), or(ii) the polypeptide sequence of wild type Citrin, isoform 2 (SEQ ID NO:3), and wherein the Citrin polypeptide has aspartate or glutamatetransport activity.

In some embodiments, the polynucleotide is single stranded. In someembodiments, the polynucleotide is double stranded. In some embodiments,the polynucleotide is DNA. In some embodiments, the polynucleotide isRNA. In some embodiments, the polynucleotide is mRNA. In someembodiments, the polynucleotide comprises at least one chemicallymodified nucleobase, sugar, backbone, or any combination thereof. Insome embodiments, the at least one chemically modified nucleobase isselected from the group consisting of pseudouracil (ψ),N1-methylpseudouracil (m1ψ), 2-thiouracil (s2U), 4′-thiouracil,5-methylcytosine, 5-methyluracil, and any combination thereof. In someembodiments, the at least one chemically modified nucleobase is selectedfrom the group consisting of pseudouracil (ψ), N1-methylpseudouracil(m1ψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4′-thiouracil,5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combinationthereof. In some embodiments, the at least one chemically modifiednucleobase is 5-methoxyuracil. In some embodiments, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 99%, or 100% of the uracils are5-methoxyuracils. In some embodiments, at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 99%, or 100% of the uracils or thymines arechemically modified. In some embodiments, at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 99%, or 100% of the guanines are chemicallymodified. In some embodiments, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 99%, or 100% of the cytosines are chemically modified. Insome embodiments, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about99%, or 100% of the adenines are chemically modified.

In some embodiments, the polynucleotide comprises at least two differentmicroRNA (miR) binding sites.

In some embodiments, the microRNA is expressed in an immune cell ofhematopoietic lineage or a cell that expresses TLR7 and/or TLR8 andsecretes pro-inflammatory cytokines and/or chemokines, and wherein thepolynucleotide (e.g., mRNA) comprises one or more modified nucleobases.

In some embodiments, the mRNA comprises at least one first microRNAbinding site of a microRNA abundant in an immune cell of hematopoieticlineage and at least one second microRNA binding site is of a microRNAabundant in endothelial cells.

In some embodiments, the mRNA comprises multiple copies of a firstmicroRNA binding site and at least one copy of a second microRNA bindingsite.

In some embodiments, the mRNA comprises first and second microRNAbinding sites of the same microRNA.

In some embodiments, the microRNA binding sites are of the 3p and 5parms of the same microRNA.

In some embodiments, the microRNA binding site comprises one or morenucleotide sequences selected from TABLE 3 or TABLE 4.

In some embodiments, the microRNA binding site binds to miR-126,miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223,miR-24, miR-27 or miR-26a, or any combination thereof.

In some embodiments, the microRNA binding site binds to miR126-3p,miR-142-3p, miR-142-5p, or miR-155, or any combination thereof.

In some embodiments, the microRNA binding site is a miR-126 bindingsite. In some embodiments, at least one microRNA binding site is amiR-142 binding site. In some embodiments, one microRNA binding site isa miR-126 binding site and the second microRNA binding site is for amicroRNA selected from the group consisting of miR-142-3p, miR-142-5p,miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24 andmiR-27.

In some embodiments, the mRNA comprises at least one miR-126-3p bindingsite and at least one miR-142-3p binding site. In some embodiments, themRNA comprises at least one miR-142-3p binding site and at least one142-5p binding site.

In some embodiments, the microRNA binding sites are located in the 5′UTR, 3′ UTR, or both the 5′ UTR and 3′ UTR of the mRNA. In someembodiments, the microRNA binding sites are located in the 3′ UTR of themRNA. In some embodiments, the microRNA binding sites are located in the5′ UTR of the mRNA. In some embodiments, the microRNA binding sites arelocated in both the 5′ UTR and 3′ UTR of the mRNA. In some embodiments,at least one microRNA binding site is located in the 3′ UTR immediatelyadjacent to the stop codon of the coding region of the mRNA. In someembodiments, at least one microRNA binding site is located in the 3′ UTR70-80 bases downstream of the stop codon of the coding region of themRNA. In some embodiments, at least one microRNA binding site is locatedin the 5′ UTR immediately preceding the start codon of the coding regionof the mRNA. In some embodiments, at least one microRNA binding site islocated in the 5′ UTR 15-20 nucleotides preceding the start codon of thecoding region of the mRNA. In some embodiments, at least one microRNAbinding site is located in the 5′ UTR 70-80 nucleotides preceding thestart codon of the coding region of the mRNA.

In some embodiments, the mRNA comprises multiple copies of the samemicroRNA binding site positioned immediately adjacent to each other orwith a spacer of less than 5, 5-10, 10-15, or 15-20 nucleotides.

In some embodiments, the mRNA comprises multiple copies of the samemicroRNA binding site located in the 3′ UTR, wherein the first microRNAbinding site is positioned immediately adjacent to the stop codon andthe second and third microRNA binding sites are positioned 30-40 basesdownstream of the 3′ most residue of the first microRNA binding site.

In some embodiments, the microRNA binding site comprises one or morenucleotide sequences selected from SEQ ID NO: 75 and SEQ ID NO: 77. Insome embodiments, the miRNA binding site binds to miR-142. In someembodiments, the miRNA binding site binds to miR-142-3p or miR-142-5p.In certain embodiments, the miR-142 comprises SEQ ID NO: 73.

In some embodiments, the microRNA binding site comprises one or morenucleotide sequences selected from SEQ ID NO:144 and SEQ ID NO:146. Insome embodiments, the miRNA binding site binds to miR-126. In someembodiments, the miRNA binding site binds to miR-126-3p or miR-126-5p.In some embodiments, the miR-126 comprises SEQ ID NO: 142.

In some embodiments, the mRNA comprises a 3′ UTR comprising a microRNAbinding site that binds to miR-142, miR-126, or a combination thereof.

In some embodiments, the polynucleotide, e.g., mRNA, further comprises a3′ UTR. In some embodiments, the 3′ UTR comprises a nucleic acidsequence at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%identical to a 3′UTR sequence selected from the group consisting of SEQID NOs: 48-72, 80, 81, 102-105, 108-117, 124, 125, 147-157, or anycombination thereof. In some embodiments, the miRNA binding site islocated within the 3′ UTR. In some embodiments, the 3′ UTR comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:48-72, 80, 81, 102-105, 108-117, 124, 125, 147-157, and any combinationthereof. In some embodiments, the mRNA comprises a 3′ UTR comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs:105, 147, 148, or any combination thereof. In some embodiments, the mRNAcomprises a 3′ UTR comprising a nucleic acid sequence of SEQ ID NO: 147.In some embodiments, the mRNA comprises a 3′ UTR comprising a nucleicacid sequence of SEQ ID NO: 148.

In some embodiments, the polynucleotide, e.g., mRNA, further comprises a5′ UTR. In some embodiments, the 5′ UTR comprises a nucleic acidsequence at least 90%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or about 100%identical to a 5′UTR sequence selected from the group consisting of SEQID NO: 30-47, 79, 120-122, 126-128, or any combination thereof. In someembodiments, the 5′ UTR comprises a sequence selected from the groupconsisting of SEQ ID NO: 30-47, 79, 120-122, 126-128, and anycombination thereof. In some embodiments, the mRNA comprises a 5′ UTRcomprising the nucleic acid sequence of SEQ ID NO: 30.

In some embodiments, the polynucleotide, e.g., mRNA, further comprises a5′ terminal cap. In some embodiments, the 5′ terminal cap comprises aCap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof. Insome embodiments, the 5′ terminal cap comprises a Cap1.

In some embodiments, the polynucleotide, e.g., mRNA, further comprises apoly-A region. In some embodiments, the poly-A region is at least about10, at least about 20, at least about 30, at least about 40, at leastabout 50, at least about 60, at least about 70, at least about 80, or atleast about 90 nucleotides in length. In some embodiments, the poly-Aregion has about 10 to about 200, about 20 to about 180, about 50 toabout 160, about 70 to about 140, about 80 to about 120 nucleotides inlength.

In some aspects, the polynucleotide, e.g., mRNA, encodes a Citrinpolypeptide that is fused to one or more heterologous polypeptides. Insome aspects, the one or more heterologous polypeptides increase apharmacokinetic property of the Citrin polypeptide. In some aspects,upon administration to a subject, the polynucleotide has (i) a longerplasma half-life; (ii) increased expression of a Citrin polypeptideencoded by the ORF; (iii) a lower frequency of arrested translationresulting in an expression fragment; (iv) greater structural stability;or (v) any combination thereof, relative to a correspondingpolynucleotide comprising SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide, e.g., mRNA, comprises (i) a5′-terminal cap; (ii) a 5′-UTR; (iii) an ORF encoding a Citrinpolypeptide; (iv) a 3′-UTR; and (v) a poly-A region. In someembodiments, the 3′-UTR comprises a miRNA binding site. In someembodiments, the polynucleotide comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 130, 133, 136, 139. Insome embodiments the polynucleotide further comprises a 5′-terminal cap(e.g., Cap1) and a poly-A-tail region (e.g., about 100 nucleotides inlength).

In some aspects, the invention relates to a method of producing apolynucleotide, e.g., mRNA, of the invention, the method comprisingmodifying an ORF encoding a Citrin polypeptide by substituting at leastone uracil nucleobase with an adenine, guanine, or cytosine nucleobase,or by substituting at least one adenine, guanine, or cytosine nucleobasewith a uracil nucleobase, wherein all the substitutions are synonymoussubstitutions. In some embodiments, the method further comprisesreplacing at least about 90%, at least about 95%, at least about 99%, orabout 100% of uracils with 5-methoxyuracils.

The present disclosure also provides a composition comprising (a) apolynucleotide, e.g., mRNA, of the invention; and (b) a delivery agent.In some embodiments, the delivery agent comprises a lipidoid, aliposome, a lipoplex, a lipid nanoparticle, a polymeric compound, apeptide, a protein, a cell, a nanoparticle mimic, a nanotube, or aconjugate. In some embodiments, the delivery agent comprises a lipidnanoparticle. In some embodiments, the lipid nanoparticle comprises alipid selected from the group consisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),(13Z,165Z)—N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608),2-({8-[(3β)-cholest-5-en-3-yloxy]octy}loxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)),(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)), and any combinations thereof. In some embodiments,the lipid nanoparticle comprises DLin-MC3-DMA.

In some aspects, the delivery agent comprises a compound having theformula (I)

or a salt or stereoisomer thereof, wherein

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,    -   —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR,        —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,        —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,        —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂,        —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(R)N(R)₂C(O)OR, and        each n is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   R₈ is selected from the group consisting of C₃₋₆ carbocycle and        heterocycle;    -   R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆        alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle        and heterocycle;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and    -   provided when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or        —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5,        or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is        1 or 2.

In some aspects, the delivery agent comprises a compound having theformula (I)

or a salt or stereoisomer thereof, wherein

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,    -   —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, and —N(R)R₈, —O(CH₂)_(n)OR,        —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,        —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,        —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂,        —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R) OR, —C(R)N(R)₂C(O)OR, and        each n is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   R₈ is selected from the group consisting of C₃₋₆ carbocycle and        heterocycle;    -   R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆        alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle        and heterocycle;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and    -   provided when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or        —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5,        or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is        1 or 2.

In some embodiments, the delivery agent comprises a compound having theformula (I), or a salt or stereoisomer thereof, wherein

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,    -   —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n        is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12,        and 13; and    -   provided when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or        —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5,        or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is        1 or 2.In some embodiments, the compound is of formula (IA)

or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5;    -   m is selected from 5, 6, 7, 8, and 9;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 1,        2, 3, 4, or 5 and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂,        —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂,        —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl, or        heterocycloalkyl;    -   M and M′ are independently selected        from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group,        and a heteroaryl group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, m is 5, 7, or 9. In some embodiments, the compoundis of Formula (IA), or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5;    -   m is selected from 5, 6, 7, 8, and 9;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 1,        2, 3, 4, or 5 and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—,    -   —P(O)(OR′)O—, an aryl group, and a heteroaryl group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, m is 5, 7, or 9.

In some embodiments, the compound is of Formula (II):

or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2,        3, or 4 and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R,        —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,        —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl, or heterocycloalkyl;    -   In some embodiments, the compound is of Formula (II), or a salt        or stereoisomer thereof, wherein    -   l is selected from 1, 2, 3, 4, and 5;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2,        3, or 4 and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—,    -   —P(O)(OR′)O—, an aryl group, and a heteroaryl group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, M₁ is M′.

In some embodiments, M and M′ are independently —C(O)O— or —OC(O)—.

In some embodiments, 1 is 1, 3, or 5.

In some embodiments, the compound is selected from the group consistingof Compound 1 to Compound 232, salts and stereoisomers thereof, and anycombination thereof.

In some embodiments, the compound is selected from the group consistingof Compound 1 to Compound 147, salts and stereoisomers thereof, and anycombination thereof.

In some aspects, the compound is of the Formula (IIa),

or a salt or stereoisomer thereof.

In some aspects, the compound is of the Formula (IIb),

or a salt or stereoisomer thereof.

In some aspects, the compound is of the Formula (IIc) or (IIe),

or a salt or stereoisomer thereof.

In some embodiments, R₄ is as described herein. In some embodiments, R₄is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.

In some aspects, the compound is of the Formula (IId),

or a salt or stereoisomer thereof,wherein n is selected from 2, 3, and 4, and m, R′, R″, and R₂ through R₆are as described herein. For example, each of R₂ and R₃ may beindependently selected from the group consisting of C₅₋₁₄ alkyl andC₅₋₁₄ alkenyl.

In some embodiments, the compound is of the Formula (IIc), or a salt orstereoisomer thereof,

wherein R₂ and R₃ are independently selected from the group consistingof C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl, n is selected from 2, 3, and 4, andR′, R″, R₅, R₆ and m are as defined herein.

In some embodiments, R₂ is C₈ alkyl.

In some embodiments, R₃ is C₅ alkyl, C₆ alkyl, C₇ alkyl, C₈ alkyl, or C₉alkyl.

In some embodiments, m is 5, 7, or 9.

In some embodiments, each R₅ is H.

In some embodiments, each R₆ is H. In some embodiments, the deliveryagent comprises a compound having the formula (III)

or salts or stereoisomers thereof, whereinring A is

t is 1 or 2;

-   -   A₁ and A₂ are each independently selected from CH or N;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   R₁, R₂, R₃, R₄, and R₅ are independently selected from the group        consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″,        and —R*OR″;    -   each M is independently selected from the group consisting        of —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—,        —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an        aryl group, and a heteroaryl group;    -   X¹, X², and X³ are independently selected from the group        consisting of a bond, —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—,        —C(O)O—, —OC(O)—, —C(O)—CH₂—, —CH₂—C(O)—, —C(O)O—CH₂—,        —OC(O)—CH₂—, —CH₂—C(O)O—, —CH₂—OC(O)—, —CH(OH)—, —C(S)—, and        —CH(SH)—;    -   each Y is independently a C₃₋₆ carbocycle;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl and a C₃₋₆ carbocycle;    -   each R′ is independently selected from the group consisting of        C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, and H; and    -   each R″ is independently selected from the group consisting of        C₃₋₁₂ alkyl and C₃₋₁₂ alkenyl,        wherein when ring A is

then

-   -   i) at least one of X¹, X², and X³ is not —CH₂—; and/or    -   ii) at least one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.

In some embodiments, the compound is of any of formulae (IIIa1)-(IIIa6):

The compounds of Formula (III) or any of (IIIa1)-(IIIa6) include one ormore of the following features when applicable.

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

wherein ring, in which the N atom is connected with X2.

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is CH.

In some embodiments, A₁ is N and A₂ is CH.

In some embodiments, A₁ is CH and A₂ is N.

In some embodiments, at least one of X¹, X², and X³ is not —CH₂—. Forexample, in certain embodiments, X¹ is not —CH₂—. In some embodiments,at least one of X¹, X², and X³ is —C(O)—.

In some embodiments, X² is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—.

In some embodiments, X³ is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—. Inother embodiments, X³ is —CH₂—.

In some embodiments, X³ is a bond or —(CH₂)₂—.

In some embodiments, R₁ and R₂ are the same. In certain embodiments, R₁,and R₃ are the same. In some embodiments, R₄ and R₅ are the same. Incertain embodiments, R₁, R₂, R₃, R₄, and R₅ are the same.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.In some embodiments, at most one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.For example, at least one of R₁, R₂, and R₃ may be —R″MR′, and/or atleast one of R₄ and R₅ is —R″MR′. In certain embodiments, at least one Mis —C(O)O—. In some embodiments, each M is —C(O)O—. In some embodiments,at least one M is —OC(O)—. In some embodiments, each M is —OC(O)—. Insome embodiments, at least one M is —OC(O)O—. In some embodiments, eachM is —OC(O)O—. In some embodiments, at least one R″ is C₃ alkyl. Incertain embodiments, each R″ is C₃ alkyl. In some embodiments, at leastone R″ is C₅ alkyl. In certain embodiments, each R″ is C₅ alkyl. In someembodiments, at least one R″ is C₆ alkyl. In certain embodiments, eachR″ is C₆ alkyl. In some embodiments, at least one R″ is C₇ alkyl. Incertain embodiments, each R″ is C₇ alkyl. In some embodiments, at leastone R′ is C₅ alkyl. In certain embodiments, each R′ is C₅ alkyl. Inother embodiments, at least one R′ is C₁ alkyl. In certain embodiments,each R′ is C₁ alkyl. In some embodiments, at least one R′ is C₂ alkyl.In certain embodiments, each R′ is C₂ alkyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is C₁₂alkyl. In certain embodiments, each of R₁, R₂, R₃, R₄, and R₅ are C₁₂alkyl.

In some embodiments, the delivery agent comprises a compound having theformula (IV)

or salts or stereoisomer thereof, wherein

-   -   A₁ and A₂ are each independently selected from CH or N and at        least one of A₁ and A₂ is N;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   R₁, R₂, R₃, R₄, and R₅ are independently selected from the group        consisting of C₆₋₂₀ alkyl and C₆₋₂₀ alkenyl;        wherein when ring A is

then

-   -   i) R₁, R₂, R₃, R₄, and R₅ are the same, wherein R₁ is not C₁₂        alkyl, C₁₈ alkyl, or C₁₈ alkenyl;    -   ii) only one of R₁, R₂, R₃, R₄, and R₅ is selected from C₆₋₂₀        alkenyl;    -   iii) at least one of R₁, R₂, R₃, R₄, and R₅ have a different        number of carbon atoms than at least one other of R₁, R₂, R₃,        R₄, and R₅;    -   iv) R₁, R₂, and R₃ are selected from C₆₋₂₀ alkenyl, and R₄ and        R₅ are selected from C₆₋₂₀ alkyl; or    -   v) R₁, R₂, and R₃ are selected from C₆₋₂₀ alkyl, and R₄ and R₅        are selected from C₆₋₂₀ alkenyl.

In some embodiments, the compound is of formula (IVa):

The compounds of Formula (IV) or (IVa) include one or more of thefollowing features when applicable.

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is CH.

In some embodiments, A₁ is N and A₂ is CH.

In some embodiments, A₁ is CH and A₂ is N.

In some embodiments, R₁, R₂, R₃, R₄, and R₅ are the same, and are notC₁₂ alkyl, C₁₈ alkyl, or C₁₈ alkenyl. In some embodiments, R₁, R₂, R₃,R₄, and R₅ are the same and are C₉ alkyl or C₁₄ alkyl.

In some embodiments, only one of R₁, R₂, R₃, R₄, and R₅ is selected fromC₆₋₂₀ alkenyl. In certain such embodiments, R₁, R₂, R₃, R₄, and R₅ havethe same number of carbon atoms. In some embodiments, R₄ is selectedfrom C₅₋₂₀ alkenyl. For example, R₄ may be C₁₂ alkenyl or C₁₈ alkenyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ have adifferent number of carbon atoms than at least one other of R₁, R₂, R₃,R₄, and R₅.

In certain embodiments, R₁, R₂, and R₃ are selected from C₆₋₂₀ alkenyl,and R₄ and R₅ are selected from C₆₋₂₀ alkyl. In other embodiments, R₁,R₂, and R₃ are selected from C₆₋₂₀ alkyl, and R₄ and R₅ are selectedfrom C₆₋₂₀ alkenyl. In some embodiments, R₁, R₂, and R₃ have the samenumber of carbon atoms, and/or R₄ and R₅ have the same number of carbonatoms. For example, R₁, R₂, and R₃, or R₄ and R₅, may have 6, 8, 9, 12,14, or 18 carbon atoms. In some embodiments, R₁, R₂, and R₃, or R₄ andR₅, are C₁₈ alkenyl (e.g., linoleyl). In some embodiments, R₁, R₂, andR₃, or R₄ and R₅, are alkyl groups including 6, 8, 9, 12, or 14 carbonatoms.

In some embodiments, R₁ has a different number of carbon atoms than R₂,R₃, R₄, and R₅. In other embodiments, R₃ has a different number ofcarbon atoms than R₁, R₂, R₄, and R₅. In further embodiments, R₄ has adifferent number of carbon atoms than R₁, R₂, R₃, and R₅.

In other embodiments, the delivery agent comprises a compound having theformula (V)

or salts or stereoisomers thereof, in which

-   -   A₃ is CH or N;    -   A₄ is CH₂ or NH; and at least one of A₃ and A₄ is N or NH;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   R₁, R₂, and R₃ are independently selected from the group        consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″,        and —R*OR″;    -   each M is independently selected        from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—,        —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl        group, and a heteroaryl group;    -   X¹ and X² are independently selected from the group consisting        of —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—,        —C(O)—CH₂—, —CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—,        —CH₂—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—;    -   each Y is independently a C₃₋₆ carbocycle;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl and a C₃₋₆ carbocycle;    -   each R′ is independently selected from the group consisting of        C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, and H; and    -   each R″ is independently selected from the group consisting of        C₃₋₁₂ alkyl and C₃₋₁₂ alkenyl.

In some embodiments, the compound is of formula (Va):

The compounds of Formula (V) or (Va) include one or more of thefollowing features when applicable.

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₃ and A₄ is N or NH.

In some embodiments, A₃ is N and A₄ is NH.

In some embodiments, A₃ is N and A₄ is CH₂.

In some embodiments, A₃ is CH and A₄ is NH.

In some embodiments, at least one of X¹ and X² is not —CH₂—. Forexample, in certain embodiments, X¹ is not —CH₂—. In some embodiments,at least one of X¹ and X² is —C(O)—.

In some embodiments, X² is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—.

In some embodiments, R₁, R₂, and R₃ are independently selected from thegroup consisting of C₅₋₂₀ alkyl and C₅₋₂₀ alkenyl. In some embodiments,R₁, R₂, and R₃ are the same. In certain embodiments, R₁, R₂, and R₃ areC₆, C₉, C₁₂, or C₁₄ alkyl. In other embodiments, R₁, R₂, and R₃ are C₁₈alkenyl. For example, R₁, R₂, and R₃ may be linoleyl.

In other embodiments, the delivery agent comprises a compound having theformula (VI):

or salts or stereoisomers thereof, in which

-   -   A₆ and A₇ are each independently selected from CH or N, wherein        at least one of A₆ and A₇ is N;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   X⁴ and X⁵ are independently selected from the group consisting        of —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—,        —C(O)—CH₂—, —CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—,        —CH₂—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—;    -   R₁, R₂, R₃, R₄, and R₅ each are independently selected from the        group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″,        —YR″, and —R*OR″;    -   each M is independently selected from the group consisting        of —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—,        —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl        group, and a heteroaryl group;    -   each Y is independently a C₃₋₆ carbocycle;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl and a C₃₋₆ carbocycle;    -   each R′ is independently selected from the group consisting of        C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, and H; and    -   each R″ is independently selected from the group consisting of        C₃₋₁₂ alkyl and C₃₋₁₂ alkenyl.

In some embodiments, R₁, R₂, R₃, R₄, and R₅ each are independentlyselected from the group consisting of C₆₋₂₀ alkyl and C₆₋₂₀ alkenyl.

In some embodiments, R₁ and R₂ are the same. In certain embodiments, R₁,R₂, and R₃ are the same. In some embodiments, R₄ and R₅ are the same. Incertain embodiments, R₁, R₂, R₃, R₄, and R₅ are the same.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is C₉₋₁₂alkyl. In certain embodiments, each of R₁, R₂, R₃, R₄, and R₅independently is C₉, C₁₂ or C₁₄ alkyl.

In certain embodiments, each of R₁, R₂, R₃, R₄, and R₅ is C₉ alkyl.

In some embodiments, A₆ is N and A₇ is N. In some embodiments, A₆ is CHand A₇ is N.

In some embodiments, X⁴ is —CH₂— and X⁵ is —C(O)—. In some embodiments,X⁴ and X⁵ are —C(O)—.

In some embodiments, when A₆ is N and A₇ is N, at least one of X⁴ and X⁵is not —CH₂—, e.g., at least one of X⁴ and X⁵ is —C(O)—. In someembodiments, when A₆ is N and A₇ is N, at least one of R₁, R₂, R₃, R₄,and R₅ is —R″MR′.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is not—R″MR′.

In some aspects, the composition is a nanoparticle composition. In someaspects, the delivery agent further comprises a phospholipid. In someembodiments, the phospholipid is selected from the group consisting of1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and any mixtures thereof.

In some embodiments, the delivery agent further comprises a structurallipid. In some embodiments, the structural lipid is selected from thegroup consisting of cholesterol, fecosterol, sitosterol, ergosterol,campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid,alpha-tocopherol, and any mixtures thereof.

In some embodiments, the delivery agent further comprises a PEG lipid.In some embodiments, the PEG lipid is selected from the group consistingof a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidicacid, a PEG-modified ceramide, a PEG-modified dialkylamine, aPEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and anymixtures thereof. In some embodiments, the PEG lipid has the formula:

wherein r is an integer between 1 and 100. In some embodiments, the PEGlipid is Compound 428.

In some aspects, the delivery agent further comprises an ionizable lipidselected from the group consisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).

In some embodiments, the delivery agent further comprises aphospholipid, a structural lipid, a PEG lipid, or any combinationthereof. In some embodiments, the delivery agent comprises Compound 18,DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of about50:10:38.5:1.5.

In some aspects, the composition is formulated for in vivo delivery. Insome aspects, the composition is formulated for intramuscular,subcutaneous, or intradermal delivery.

The present disclosure further provides a polynucleotide comprising anmRNA comprising: (i) a 5′ UTR, (ii) an open reading frame (ORF) encodinga human Citrin polypeptide, wherein the ORF comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs 5-29, 129,132, 135, 138, and (iii) a 3′ UTR comprising a microRNA binding siteselected from miR-142, miR-126, or a combination thereof, wherein themRNA comprises at least one chemically modified nucleobase.

The present disclosure further provides a polynucleotide comprising anmRNA comprising: (i) a 5′-terminal cap; (ii) a 5′ UTR comprising asequence selected from the group consisting of SEQ ID NO: 30-47, 79,120-122, 126-128, and any combination thereof; (iii) an open readingframe (ORF) encoding a human Citrin polypeptide, wherein the ORFcomprises a sequence selected from the group consisting of SEQ ID NOs:5-29, 129, 132, 135, 138, wherein the mRNA comprises at least onechemically modified nucleobase selected from the group consisting ofpseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil,2-thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil,5-methoxyuracil, and any combination thereof and (iv) a 3′ UTRcomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 48-72, 80, 81, 102-105, 108-117, 124, 125, 147-157, and anycombination thereof; and (v) a poly-A-region.

In some embodiments, the polynucleotide comprises a nucleic acidsequence selected from the group consisting of SEQ ID NO: 130, 133, 136,139.

The present disclosure further provides a pharmaceutical compositioncomprising the polynucleotide, e.g., an mRNA, and a delivery agent. Insome embodiments, the delivery agent is a lipid nanoparticle comprisingCompound 18, Compound 236, a salt or a stereoisomer thereof, or anycombination thereof. In some embodiments, the polynucleotide comprisinga nucleotide sequence encoding a Citrin polypeptide disclosed herein isformulated with a delivery agent comprising, e.g., a compound having theFormula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compoundhaving the Formula (III), (IV), (V), or (VI), e.g., any of Compounds233-342, e.g., Compound 236; or a compound having the Formula (VIII),e.g., any of Compounds 419-428, e.g., Compound 428, or any combinationthereof. In some embodiments, the delivery agent comprises Compound 18,DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of about50:10:38.5:1.5.

In one aspect of the embodiments disclosed herein, the subject is ahuman subject in need of treatment or prophylaxis for Citrullinemia Type2 (CTLN2).

In one aspect of the embodiments disclosed herein, upon administrationto the subject, the mRNA has: (i) a longer plasma half-life; (ii)increased expression of a Citrin polypeptide encoded by the ORF; (iii) alower frequency of arrested translation resulting in an expressionfragment; (iv) greater structural stability; or (v) any combinationthereof, relative to a corresponding mRNA having the nucleic acidsequence of SEQ ID NO: 2 or 4 and/or administered as naked mRNA.

In some embodiments, a pharmaceutical composition or polynucleotidedisclosed herein is suitable for administration as a single unit dose ora plurality of single unit doses.

In some embodiments, a pharmaceutical composition or polynucleotidedisclosed herein is suitable for reducing the level of one or morebiomarkers of CTLN2 in the subject.

In some embodiments, a pharmaceutical composition or polynucleotidedisclosed herein is for use in treating, preventing or delaying theonset of CTLN2 signs or symptoms in the subject. In some embodiments,the signs or symptoms include nocturnal delirium, aggression,irritability, hyperactivity, delusions, disorientation, restlessness,drowsiness, loss of memory, flapping tremor, convulsive seizures, coma,brain edema, death, or a combination thereof.

In some aspects, the invention relates to a host cell comprising apolynucleotide of the invention. In some aspects, the host cell is aeukaryotic cell.

In some aspects, the invention relates to a vector comprising apolynucleotide of the invention.

In some aspects, the invention relates to a method of making apolynucleotide comprising enzymatically or chemically synthesizing apolynucleotide of the invention.

In some aspects, the invention relates to a method of expressing in vivoan active Citrin polypeptide in a subject in need thereof comprisingadministering to the subject an effective amount of a polynucleotide,composition, host cell, or vector of the invention.

In some aspects, the invention relates to a method of treatingcitrullinemia type 2 (CTLN2) in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of apolynucleotide, composition, host cell, or vector of the invention,wherein the administration alleviates the signs or symptoms of CTLN2 inthe subject.

In some aspects, the invention relates to a method to prevent or delaythe onset of CTLN2 signs or symptoms in a subject in need thereofcomprising administering to the subject a prophylactically effectiveamount of a polynucleotide, composition, host cell, or vector of theinvention before CTLN2 signs or symptoms manifest, wherein theadministration prevents or delays the onset of CTLN2 signs or symptomsin the subject.

In some aspects, the invention relates to a method to ameliorate thesigns or symptoms of CTLN2 in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of apolynucleotide, composition, host cell, or vector of the inventionbefore CTLN2 signs or symptoms manifest, wherein the administrationameliorates CTLN2 signs or symptoms in the subject.

The present disclosure further provides a method of expressing a Citrinpolypeptide in a human subject in need thereof comprising administeringto the subject an effective amount of a pharmaceutical composition or apolynucleotide, e.g., an mRNA, described herein, wherein thepharmaceutical composition or polynucleotide is suitable foradministrating as a single dose or as a plurality of single unit dosesto the subject.

The present disclosure further provides a method of treating, preventingor delaying the onset of Citrullinemia Type 2 (CTLN2) signs or symptomsin a human subject in need thereof comprising administering to thesubject an effective amount of a pharmaceutical composition or apolynucleotide, e.g., an mRNA, described herein, wherein theadministration treats, prevents or delays the onset of one or more ofthe signs or symptoms of CTLN2 in the subject.

The present disclosure further provides a method for the treatment ofCitrullinemia Type 2 (CTLN2), comprising administering to a humansubject suffering from CTLN2 a single intravenous dose of apharmaceutical composition or a polynucleotide, e.g., an mRNA, describedherein.

The present disclosure further provides a method of reducing an ammoniaand/or triglyceride blood and/or plasma level in a human subjectcomprising administering to the subject an effective amount of apharmaceutical composition or a polynucleotide, e.g., an mRNA, describedherein, wherein the administration reduces the ammonia and/ortriglyceride plasma level in the subject. In some embodiments,

-   -   (i) ammonia blood and/or plasma level is reduced at least        2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at        least 15 fold, at least 20-fold, at least 50-fold, at least 20%,        at least 30%, at least 40%, at least 50%, at least 60%, at least        70%, at least 80%, at least 90%, at least 95% or at least 98%,        at least 99%, or 100% compared to the subject's baseline level        or a reference ammonia blood and/or plasma level, for at least        24 hours, at least 48 hours, at least 72 hours, at least 96        hours, or at least 120 hours post-administration; and/or    -   (ii) triglyceride blood and/or plasma level is reduced at least        1.5-fold, 2-fold, at least 3-fold, at least 4-fold, at least        5-fold, at least 10-fold, at least 20-fold, at least 20%, at        least 30%, at least 40%, at least 50%, at least 60%, at least        70%, at least 80%, at least 90%, at least 95% or at least 98%,        at least 99%, or 100% compared to the subject's baseline level        or a reference triglyceride blood and/or plasma level, for at        least 24 hours, at least 48 hours, at least 72 hours, at least        96 hours, or at least 120 hours post-administration.

In some embodiments, after administration of the pharmaceuticalcomposition or polynucleotide to the subject, e.g., within 12 hours, 24hours, 36 hours, or 48 hours, the Citrin activity in the subject isincreased at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 150%, at least 200%, at least 300%, at least 400%, at least 500%,or at least 600% compared to the subject's baseline Citrin activity.

In some embodiments, the Citrin activity is increased in the liver,and/or kidneys of the subject.

In some embodiments, the increased Citrin activity persists for greaterthan 24, 36, 48, 60, 72, or 96 hours post-administration.

In some embodiments, the pharmaceutical composition or polynucleotide isadministered to the subject having citrullinemia type 2 (CTLN2).

In some embodiments, after administration of the pharmaceuticalcomposition or polynucleotide to the subject, e.g., within 24 hours, thelevel of ammonia in the subject is reduced by at least about 100%, atleast about 90%, at least about 80%, at least about 70%, at least about60%, at least about 50%, at least about 40%, or at least about 30%compared to the subject's baseline ammonia level.

In some embodiments, the level of ammonia is reduced in the blood and/orplasma of the subject.

In some embodiments, after the administration to the subject the levelof ammonia in the subject is reduced compared to the baseline level inthe subject for at least one day, at least two days, at least threedays, at least four days, at least five days, at least one week, atleast two weeks, at least three weeks, or at least one month.

In some embodiments, after administration of the pharmaceuticalcomposition or polynucleotide to the subject, e.g., within 24 hours, thelevel of triglycerides in the subject is reduced by at least about 100%,at least about 80%, at least about 60%, at least about 50%, at leastabout 40%, at least about 30%, at least about 20%, or at least about 10%compared to the subject's baseline triglycerides.

In some embodiments, the level of triglycerides is reduced in the plasmaand/or liver of the subject.

In some embodiments, after the administration to the subject the levelof triglycerides in the subject is reduced compared to the baselinelevel in the subject for at least one day, at least two days, at leastthree days, at least four days, at least five days, at least one week,at least two weeks, at least three weeks, or at least one month.

In some embodiments, the subject is symptomatic for CTLN2.

In some embodiments, the subject is presymptomatic for CTLN2.

In some embodiments, the Citrin polypeptide activity level is sufficientto prevent the onset of CTLN2 signs and/or symptoms.

In some embodiments, the pharmaceutical composition or polynucleotide isadministered as a single dose of less than 1.5 mg/kg, less than 1.25mg/kg, less than 1 mg/kg, or less than 0.75 mg/kg.

In some embodiments, the administration to the subject is about once aweek, about once every two weeks, or about once a month.

In some embodiments, the pharmaceutical composition or polynucleotide isadministered intravenously.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-1D show the protein sequence (FIG. 1A), table with domainfeatures (panel B), graphic representation of domain structure (FIG.1C), and nucleic acid sequence (FIG. 1D) of isoform 1 of Citrin.

FIGS. 2A-2D show the protein sequence (FIG. 2A), table with domainfeatures (FIG. 2B), graphic representation of domain structure (FIG.2C), and nucleic acid sequence (FIG. 2D) of isoform 2 of Citrin.

FIG. 3 shows uracil (U) metrics corresponding to wild type isoform 1 ofCitrin and 25 sequence optimized Citrin polynucleotides. The columnlabeled “U content (%)” corresponds to the % Um parameter. The columnlabeled “U Content v. WT (%)” corresponds to % U_(WT). The columnlabeled “U Content v. Theoretical Minimum (%)” corresponds to % U_(TM).The column labeled “UU pairs v. WT (%)” corresponds to % UU_(WT).

FIG. 4 shows guanine (G) metrics corresponding to wild type isoform 1 ofCitrin and 25 sequence optimized Citrin polynucleotides. The columnlabeled “G Content (%)” corresponds to % Gm. The column labeled “GContent v. WT (%)” corresponds to % G_(WT). The column labeled “GContent v. Theoretical Maximum (%)” corresponds to % G_(TMX).

FIG. 5 shows cytosine (C) metrics corresponding to wild type isoform 1of Citrin and 25 sequence optimized Citrin polynucleotides. The columnlabeled “C Content (%)” corresponds to % Cm. The column labeled “CContent v. WT (%)” corresponds to % C_(WT). The column labeled “CContent v. Theoretical Maximum (%)” corresponds to % C_(TMX).

FIG. 6 shows guanine plus cytosine (G/C) metrics corresponding to wildtype isoform 1 of Citrin and 25 sequence optimized Citrinpolynucleotides. The column labeled “G/C Content (%)” corresponds to %G/Cm. The column labeled “G/C Content v. WT (%)” corresponds to % G/CWT.The column labeled “G/C Content v. Theoretical Maximum (%)” correspondsto % G/C_(TMX).

FIG. 7 is an experimental design diagram showing the timing foradministration of mRNA encoding Citrin or non-translated Factor IX(NTFIX) control mRNA to CTRN knockout and mGPD/CTRN double knockout miceand the timing for bleeding mice, taking body weight measurements, andcollecting samples (plasma, liver, and spleen).

FIG. 8 shows blood ammonium levels (μg/dl) in CTRN knockout (Ctrn KO)and mGPD/CTRN (Double KO) mice administered mRNA (Construct #1) encodinghuman Citrin (hCitrin) or control mRNA encoding non-translated Factor IX(NtFix) according to the experimental design shown in FIG. 7. Bloodammonium levels for wild-type mice administered control NTFIX mRNA(WT-NtFix) are also shown.

FIGS. 9A-9B show sucrose aversion (preference index) before mRNAadministration (FIG. 9A) and after the second injection of mRNA (FIG.9B) for mGPD/CTRN double knockout mice administered mRNA (Construct #1)encoding Citrin or control NTFIX mRNA.

FIG. 10 shows protein expression levels of Citrin (top panel), mGPD(middle panel), and citrate synthase (bottom panel) in isolated livermitochondria following administration of control mRNA (NTFIX) or mRNAencoding Citrin (Construct #1) to wild-type mice, CTRN knockout mice,and mGPD/CTRN knockout mice.

FIG. 11 shows a semi-quantitative analysis of protein expression levelsof Citrin following administration of control mRNA (NTFIX) or mRNAencoding Citrin (Construct #1) to wild-type mice, CTRN knockout mice,and mGPD/CTRN knockout mice.

FIG. 12 is an experimental design diagram showing the timing of mRNAinjection and sucrose challenge for the study described in Example 23.

FIGS. 13A-13B show plasma ammonia concentration levels in mGPD/CTRNdouble knockout mice after injection of mRNA encoding human Citrin (0.5mg/kg and 0.2 mg/kg) or control mRNA (NTFIX). FIG. 13A shows the plasmaammonia concentration prior to injection of mRNA and 24 hours postinitial injection of mRNA. FIG. 13B shows the plasma ammoniaconcentration 16 days post initial injection of mRNA. FIGS. 14A-14B showCitrin protein expression in liver mitochondria isolated from miceadministered Citrin mRNA. FIG. 14A is a Western blot showing Citrinprotein expression in liver mitochondria isolated from wild-type mice 24hours post-injection with mRNA encoding human Citrin (Constructs #1, #2,#3 or #4) or with mRNA encoding NTFIX. α-tubulin is detected as aloading control. FIG. 14B shows a quantitative analysis of the ratio ofprotein expression levels of Citrin in liver mitochondria isolated fromwild-type mice 24 hours post-injection with mRNA encoding Citrin(Constructs #1, 2, 3 or 4) to control mRNA (NTFIX).

DETAILED DESCRIPTION

The present invention provides mRNA therapeutics for the treatment ofcitrullinemia type 2 (CTLN2). CTLN2 is an autosomal recessive urea cycledisorder that causes abnormal buildup of ammonia and other toxicsubstances in the blood. CTLN2 is a form of citrullinemia that is causedby mutation of the Solute Carrier Family 25, Member 13 gene(“SLC25A13”). CTLN2 is also known as adult-onset citrullinemia type 2 orcitrin deficiency. CTLN2 causes nocturnal delirium, aggression,irritability, hyperactivity, delusions, disorientation, restlessness,drowsiness, loss of memory, flapping tremor, convulsive seizures, coma,or even death resulting from brain edema. mRNA therapeutics areparticularly well-suited for the treatment of CTLN2 as the technologyprovides for the intracellular delivery of mRNA encoding Citrin (theprotein encoded by SLC25A13) followed by de novo synthesis of functionalCitrin protein within target cells. After delivery of mRNA to the targetcells, the desired Citrin protein is expressed by the cells' owntranslational machinery, and hence, fully functional Citrin proteinreplaces the defective or missing protein.

One challenge associated with delivering nucleic acid-based therapeutics(e.g., mRNA therapeutics) in vivo stems from the innate immune responsewhich can occur when the body's immune system encounters foreign nucleicacids. Foreign mRNAs can activate the immune system via recognitionthrough toll-like receptors (TLRs), in particular TLR7/8, which isactivated by single-stranded RNA (ssRNA). In nonimmune cells, therecognition of foreign mRNA can occur through the retinoicacid-inducible gene I (RIG-I). Immune recognition of foreign mRNAs canresult in unwanted cytokine effects including interleukin-1β (IL-1β)production, tumor necrosis factor-α (TNF-α) distribution and a strongtype I interferon (type I IFN) response. The instant invention featuresthe incorporation of different modified nucleotides within therapeuticmRNAs to minimize the immune activation and optimize the translationefficiency of mRNA to protein. Particular aspects of the inventionfeature a combination of nucleotide modification to reduce the innateimmune response and sequence optimization, in particular, within theopen reading frame (ORF) of therapeutic mRNAs encoding Citrin to enhanceprotein expression.

Certain embodiments of the mRNA therapeutic technology of the instantinvention also features delivery of mRNA encoding Citrin via a lipidnanoparticle (LNP) delivery system. Lipid nanoparticles (LNPs) are anideal platform for the safe and effective delivery of mRNAs to targetcells. LNPs have the unique ability to deliver nucleic acids by amechanism involving cellular uptake, intracellular transport andendosomal release or endosomal escape. The instant invention featuresnovel ionizable lipid-based LNPs combined with mRNA encoding Citrinwhich have improved properties when administered in vivo. Without beingbound in theory, it is believed that the novel ionizable lipid-based LNPformulations of the invention have improved properties, for example,cellular uptake, intracellular transport and/or endosomal release orendosomal escape. LNPs administered by systemic route (e.g., intravenous(IV) administration), for example, in a first administration, canaccelerate the clearance of subsequently injected LNPs, for example, infurther administrations. This phenomenon is known as accelerated bloodclearance (ABC) and is a key challenge, in particular, when replacingdeficient proteins (e.g., Citrin) in a therapeutic context. This isbecause repeat administration of mRNA therapeutics is in most instancesessential to maintain necessary levels of protein in target tissues insubjects (e.g., subjects suffering from CTLN2.) Repeat dosing challengescan be addressed on multiple levels. mRNA engineering and/or efficientdelivery by LNPs can result in increased levels and or enhanced durationof protein (e.g., Citrin) being expressed following a first dose ofadministration, which in turn, can lengthen the time between first doseand subsequent dosing. It is known that the ABC phenomenon is, at leastin part, transient in nature, with the immune responses underlying ABCresolving after sufficient time following systemic administration. Assuch, increasing the duration of protein expression and/or activityfollowing systemic delivery of an mRNA therapeutic of the invention inone aspect, combats the ABC phenomenon. Moreover, LNPs can be engineeredto avoid immune sensing and/or recognition and can thus further avoidABC upon subsequent or repeat dosing. Exemplary aspect of the inventionfeature novel LNPs which have been engineered to have reduced ABC.

1. Citrin

The principal gene associated with CTLN2 is SLC25A13, which has twovariants (Genbank Accession Nos. NM_001160210, NP_001153682; NM_014251,NP_055066; XM_006715831, XP_006715894; XM_011515727, XP_011514029).SLC25A13 encodes Citrin, which is a calcium-dependent mitochondrialsolute carrier protein. Citrin is also known as Mitochondrial AspartateGlutamate Carrier 2 or ARALAR2. Citrin plays a critical role in the ureacycle by catalyzing the mitochondrial uptake of glutamate and export ofaspartate. Saheki, T. et al., Metab. Brain Dis. 17:335-346 (2002).Depending on the variant, the precursor form of human Citrin is either676 or 675 amino acids, while its mature form is either 675 or 674 aminoacids with the initiator methionine cleaved off.

In certain aspects, the invention provides a polynucleotide (e.g., aribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprising anucleotide sequence (e.g., an open reading frame (ORF)) encoding aCitrin polypeptide. In some embodiments, the Citrin polypeptide of theinvention is a wild type Citrin isoform 1 or 2 protein. In someembodiments, the Citrin polypeptide of the invention is a variant, apeptide or a polypeptide containing a substitution, and insertion and/oran addition, a deletion and/or a covalent modification with respect to awild-type Citrin isoform 1 or 2 sequence. In some embodiments, sequencetags or amino acids, can be added to the sequences encoded by thepolynucleotides of the invention (e.g., at the N-terminal or C-terminalends), e.g., for localization. In some embodiments, amino acid residueslocated at the carboxy, amino terminal, or internal regions of apolypeptide of the invention can optionally be deleted providing forfragments.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA)comprising a nucleotide sequence (e.g., an ORF) of the invention encodesa substitutional variant of a Citrin isoform 1 or 2 sequence, which cancomprise one, two, three or more than three substitutions. In someembodiments, the substitutional variant can comprise one or moreconservative amino acids substitutions. In other embodiments, thevariant is an insertional variant. In other embodiments, the variant isa deletional variant.

As recognized by those skilled in the art, Citrin isoform 1 or 2 proteinfragments, functional protein domains, variants, and homologous proteins(orthologs) are also considered to be within the scope of the Citrinpolypeptides of the invention. Nonlimiting examples of polypeptidesencoded by the polynucleotides of the invention are shown in FIGS. 1 and2. For example, FIG. 1 shows the amino acid sequence of human Citrinwild type isoform 1.

Certain compositions and methods presented in this disclosure refer tothe protein or polynucleotide sequences of Citrin isoform 1. A personskilled in the art will understand that such disclosures are equallyapplicable to any other isoforms of Citrin known in the art.

2. Polynucleotides and Open Reading Frames (ORFs)

The instant invention features mRNAs for use in treating (i.e.,prophylactically and/or therapeutically treating) CTLN2. The mRNAsfeatured for use in the invention are administered to subjects andencode Citrin proteins(s) in vivo. Accordingly, the invention relates topolynucleotides comprising an open reading frame of linked nucleosidesencoding human Citrin, isoforms thereof, functional fragments thereof,and fusion proteins comprising Citrin. In some embodiments, the openreading frame is sequence-optimized. In particular embodiments, theinvention provides sequence-optimized polynucleotides comprisingnucleotides encoding the polypeptide sequence of isoforms 1 or 2 ofhuman Citrin, or sequence having high sequence identity with thosesequence optimized polynucleotides.

In certain aspects, the invention provides polynucleotides (e.g., a RNA,e.g., an mRNA) that comprise a nucleotide sequence (e.g., an ORF)encoding one or more Citrin polypeptides. In some embodiments, theencoded Citrin polypeptide of the invention can be selected from:

-   -   (i) a full length Citrin polypeptide (e.g., having the same or        essentially the same length as wild-type Citrin isoform 1 or 2);    -   (ii) a functional fragment of any of the Citrin isoforms        described herein (e.g., a truncated (e.g., deletion of carboxy,        amino terminal, or internal regions) sequence shorter than one        of wild-type isoforms 1 or 2; but still retaining Citrin        enzymatic activity);    -   (iii) a variant thereof (e.g., full length or truncated isoform        1 or 2 proteins in which one or more amino acids have been        replaced, e.g., variants that retain all or most of the Citrin        activity of the polypeptide with respect to a reference isoform        (such as, e.g., any natural or artificial variants known in the        art); or    -   (iv) a fusion protein comprising (i) a full length Citrin        isoform 1 or 2 protein, a functional fragment or a variant        thereof, and (ii) a heterologous protein.

In certain embodiments, the encoded Citrin polypeptide is a mammalianCitrin polypeptide, such as a human Citrin polypeptide, a functionalfragment or a variant thereof.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention increases Citrin protein expression levels and/ordetectable Citrin enzymatic activity levels in cells when introduced inthose cells, e.g., by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100%, compared to Citrin protein expression levels and/or detectableCitrin enzymatic activity levels in the cells prior to theadministration of the polynucleotide of the invention. Citrin proteinexpression levels and/or Citrin enzymatic activity can be measuredaccording to methods know in the art. In some embodiments, thepolynucleotide is introduced to the cells in vitro. In some embodiments,the polynucleotide is introduced to the cells in vivo.

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) that encodesa wild-type human Citrin, e.g., wild-type isoform 1 of human Citrin (SEQID NO: 1, see FIG. 1), or wild-type isoform 2 of human Citrin (SEQ IDNO: 3, see FIG. 2).

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a codon optimized nucleic acid sequence, whereinthe open reading frame (ORF) of the codon optimized nucleic acidsequence is derived from a wild-type Citrin sequence (e.g., wild-typeisoforms 1 or 2). For example, for polynucleotides of inventioncomprising a sequence optimized ORF encoding Citrin isoform 2, thecorresponding wild type sequence is the native Citrin isoform 2.Similarly, for an sequence optimized mRNA encoding a functional fragmentof isoform 1, the corresponding wild type sequence is the correspondingfragment from Citrin isoform 1.

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence encoding Citrin isoform 1having the full length sequence of human Citrin isoform 1 (i.e.,including the initiator methionine). In mature human Citrin isoform 1,the initiator methionine can be removed to yield a “mature Citrin”comprising amino acid residues of 2-606 of the translated product. Theteachings of the present disclosure directed to the full sequence ofhuman Citrin (amino acids 1-606) are also applicable to the mature formof human Citrin lacking the initiator methionine (amino acids 2-606).Thus, in some embodiments, the polynucleotides (e.g., a RNA, e.g., anmRNA) of the invention comprise a nucleotide sequence encoding Citrinisoform 1 having the mature sequence of human Citrin isoform 1 (i.e.,lacking the initiator methionine). In some embodiments, thepolynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprisinga nucleotide sequence encoding Citrin isoform 1 having the full lengthor mature sequence of human Citrin isoform 1 is sequence optimized.

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) encoding amutant Citrin polypeptide. In some embodiments, the polynucleotides ofthe invention comprise an ORF encoding a Citrin polypeptide thatcomprises at least one point mutation in the Citrin sequence and retainsCitrin enzymatic activity. In some embodiments, the mutant Citrinpolypeptide has a Citrin activity which is at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100% of the Citrin activity of the correspondingwild-type Citrin (i.e., the same Citrin isoform but without themutation(s)). In some embodiments, the polynucleotide (e.g., a RNA,e.g., an mRNA) of the invention comprising an ORF encoding a mutantCitrin polypeptide is sequence optimized.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) thatencodes a Citrin polypeptide with mutations that do not alter Citrinenzymatic activity. Such mutant Citrin polypeptides can be referred toas function-neutral. In some embodiments, the polynucleotide comprisesan ORF that encodes a mutant Citrin polypeptide comprising one or morefunction-neutral point mutations.

In some embodiments, the mutant Citrin polypeptide has higher Citrinenzymatic activity than the corresponding wild-type Citrin. In someembodiments, the mutant Citrin polypeptide has a Citrin activity that isat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100% higher than theactivity of the corresponding wild-type Citrin (i.e., the same Citrinisoform but without the mutation(s)).

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) encoding afunctional Citrin fragment, e.g., where one or more fragments correspondto a polypeptide subsequence of a wild type Citrin polypeptide andretain Citrin enzymatic activity. In some embodiments, the Citrinfragment has a Citrin activity which is at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100% of the Citrin activity of the correspondingfull length Citrin. In some embodiments, the polynucleotides (e.g., aRNA, e.g., an mRNA) of the invention comprising an ORF encoding afunctional Citrin fragment is sequence optimized.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin fragment that has higher Citrin enzymatic activity than thecorresponding full length Citrin. Thus, in some embodiments the Citrinfragment has a Citrin activity which is at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100% higher than the Citrin activity of thecorresponding full length Citrin.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin fragment that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24% or 25% shorter than wild-type isoform 1 or 2 of Citrin.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof), wherein the nucleotide sequence is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto the sequence of SEQ ID NO:2 or 4 (see, e.g., panel D in FIGS. 1 and2, respectively).

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof), wherein the nucleotide sequence has at least 70%,at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to a sequence selected from the group consisting of SEQ ID NOs:5-29, 129, 132, 135, and 138. See TABLE 2.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof), wherein the nucleotide sequence has 70% to 100%,75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to90%, 90% to 95%, or 95% to 100%, sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 5-29, 129, 132, 135,and 138. See TABLE 2.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises an ORF encoding a Citrin polypeptide (e.g., thewild-type sequence, functional fragment, or variant thereof), whereinthe polynucleotide comprises a nucleic acid sequence having 70% to 100%,75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to90%, 90% to 95%, or 95% to 100%, sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 130, 133, 136, and139. See TABLE 5.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof), wherein the nucleotide sequence is at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to thesequence of SEQ ID NO:2 or 4 (see, e.g., panel D in FIGS. 1 and 2,respectively).

In some embodiments the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof), wherein the nucleotide sequence is between 70% and90% identical; between 71% and 85% identical; between 72% and 84%identical; between 73% and 82% identical, between 73% and 80% identical,between 73% and 78%, or between 73% and 76% identical to the sequence ofSEQ ID NO:2 or 4 (see, e.g., panel D in FIGS. 1 and 2, respectively).

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises from about 900 to about 100,000 nucleotides(e.g., from 900 to 1,000, from 900 to 1,100, from 900 to 1,200, from 900to 1,300, from 900 to 1,400, from 900 to 1,500, from 1,000 to 1,100,from 1,000 to 1,100, from 1,000 to 1,200, from 1,000 to 1,300, from1,000 to 1,400, from 1,000 to 1,500, from 1,083 to 1,200, from 1,083 to1,400, from 1,083 to 1,600, from 1,083 to 1,800, from 1,083 to 2,000,from 1,083 to 3,000, from 1,083 to 5,000, from 1,083 to 7,000, from1,083 to 10,000, from 1,083 to 25,000, from 1,083 to 50,000, from 1,083to 70,000, or from 1,083 to 100,000).

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof), wherein the length of the nucleotide sequence(e.g., an ORF) is at least 500 nucleotides in length (e.g., at least orgreater than about 500, 600, 700, 80, 900, 1,000, 1,050, 1,083, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100,4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100,5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 7,000,8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,80,000, 90,000 or up to and including 100,000 nucleotides).

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof) further comprises at least one nucleic acid sequencethat is noncoding, e.g., a miRNA binding site. In some embodiments, thepolynucleotide (e.g., a RNA, e.g., an mRNA) of the invention furthercomprises a 5′-UTR (e.g., selected from the sequences of SEQ ID NOs:30-47, 79, 120-122, 126-128) and a 3′UTR (e.g., selected from thesequences of SEQ ID NOs: 48-72, 80, 81, 102-105, 108-117, 124, 125,147-157). In some embodiments, the polynucleotide (e.g., a RNA, e.g., anmRNA) of the invention comprises a sequence selected from the groupconsisting of SEQ ID NO: 5-29, 129, 132, 135, and 138. In a furtherembodiment, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a5′ terminal cap (e.g., Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′methylG cap, or an analog thereof) and a poly-A-tail region (e.g., about100 nucleotides in length). In a further embodiment, the polynucleotide(e.g., a RNA, e.g., an mRNA) a comprises a 3′ UTR comprising a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 105,147, 148, or any combination thereof. In a further embodiment, thepolynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 3′ UTRcomprising a nucleic acid sequence of SEQ ID NO: 147. In a furtherembodiment, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a3′ UTR comprising a nucleic acid sequence of SEQ ID NO: 148.

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding aCitrin polypeptide is single stranded or double stranded.

In some embodiments, the polynucleotide of the invention comprising anucleotide sequence (e.g., an ORF) encoding a Citrin polypeptide (e.g.,the wild-type sequence, functional fragment, or variant thereof) is DNAor RNA. In some embodiments, the polynucleotide of the invention is RNA.In some embodiments, the polynucleotide of the invention is, orfunctions as, a messenger RNA (mRNA). In some embodiments, the mRNAcomprises a nucleotide sequence (e.g., an ORF) that encodes at least oneCitrin polypeptide, and is capable of being translated to produce theencoded Citrin polypeptide in vitro, in vivo, in situ or ex vivo.

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprises a sequence-optimized nucleotide sequence (e.g.,an ORF) encoding a Citrin polypeptide (e.g., the wild-type sequence,functional fragment, or variant thereof), wherein the polynucleotidecomprises at least one chemically modified nucleobase, e.g.,5-methoxyuracil. In some embodiments, the polynucleotide furthercomprises a miRNA binding site, e.g., a miRNA binding site that binds tomiR-142 and/or a miRNA binding site that binds to miR-126. In someembodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) disclosedherein is formulated with a delivery agent comprising, e.g., a compoundhaving the Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18;a compound having the Formula (III), (IV), (V), or (VI), e.g., any ofCompounds 233-342, e.g., Compound 236; or a compound having the Formula(VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or anycombination thereof. In some embodiments, the delivery agent comprisesCompound 18, DSPC, Cholesterol, and Compound 428, e.g., with a moleratio of about 50:10:38.5:1.5.

3. Signal Sequences

The polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention canalso comprise nucleotide sequences that encode additional features thatfacilitate trafficking of the encoded polypeptides to therapeuticallyrelevant sites. One such feature that aids in protein trafficking is thesignal sequence, or targeting sequence. The peptides encoded by thesesignal sequences are known by a variety of names, including targetingpeptides, transit peptides, and signal peptides. In some embodiments,the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotidesequence (e.g., an ORF) that encodes a signal peptide operably linked toa nucleotide sequence that encodes a Citrin polypeptide describedherein.

In some embodiments, the “signal sequence” or “signal peptide” is apolynucleotide or polypeptide, respectively, which is from about 9 to200 nucleotides (3-70 amino acids) in length that, optionally, isincorporated at the 5′ (or N-terminus) of the coding region or thepolypeptide, respectively. Addition of these sequences results intrafficking the encoded polypeptide to a desired site, such as theendoplasmic reticulum or the mitochondria through one or more targetingpathways. Some signal peptides are cleaved from the protein, for exampleby a signal peptidase after the proteins are transported to the desiredsite.

In some embodiments, the polynucleotide of the invention comprises anucleotide sequence encoding a Citrin polypeptide, wherein thenucleotide sequence further comprises a 5′ nucleic acid sequenceencoding a heterologous signal peptide.

4. Fusion Proteins

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) can comprise more than one nucleic acid sequence (e.g.,an ORF) encoding a polypeptide of interest. In some embodiments,polynucleotides of the invention comprise a single ORF encoding a Citrinpolypeptide, a functional fragment, or a variant thereof. However, insome embodiments, the polynucleotide of the invention can comprise morethan one ORF, for example, a first ORF encoding a Citrin polypeptide (afirst polypeptide of interest), a functional fragment, or a variantthereof, and a second ORF expressing a second polypeptide of interest.In some embodiments, two or more polypeptides of interest can begenetically fused, i.e., two or more polypeptides can be encoded by thesame ORF. In some embodiments, the polynucleotide can comprise a nucleicacid sequence encoding a linker (e.g., a G₄S peptide linker or anotherlinker known in the art) between two or more polypeptides of interest.

In some embodiments, a polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) can comprise two, three, four, or more ORFs, eachexpressing a polypeptide of interest.

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) can comprise a first nucleic acid sequence (e.g., a firstORF) encoding a Citrin polypeptide and a second nucleic acid sequence(e.g., a second ORF) encoding a second polypeptide of interest.

5. Sequence Optimization of Nucleotide Sequence Encoding a CitrinPolypeptide

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention is sequence optimized. In some embodiments, thepolynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises anucleotide sequence (e.g., an ORF) encoding a Citrin polypeptide, anucleotide sequence (e.g., an ORF) encoding another polypeptide ofinterest, a 5′-UTR, a 3′-UTR, a miRNA, a nucleotide sequence encoding alinker, or any combination thereof) that is sequence optimized.

A sequence-optimized nucleotide sequence, e.g., an codon-optimized mRNAsequence encoding a Citrin polypeptide, is a sequence comprising atleast one synonymous nucleobase substitution with respect to a referencesequence (e.g., a wild type nucleotide sequence encoding a Citrinpolypeptide).

A sequence-optimized nucleotide sequence can be partially or completelydifferent in sequence from the reference sequence. For example, areference sequence encoding polyserine uniformly encoded by TCT codonscan be sequence-optimized by having 100% of its nucleobases substituted(for each codon, Tin position 1 replaced by A, C in position 2 replacedby G, and T in position 3 replaced by C) to yield a sequence encodingpolyserine which would be uniformly encoded by AGC codons. Thepercentage of sequence identity obtained from a global pairwisealignment between the reference polyserine nucleic acid sequence and thesequence-optimized polyserine nucleic acid sequence would be 0%.However, the protein products from both sequences would be 100%identical.

Some sequence optimization (also sometimes referred to codonoptimization) methods are known in the art (and discussed in more detailbelow) and can be useful to achieve one or more desired results. Theseresults can include, e.g., matching codon frequencies in certain tissuetargets and/or host organisms to ensure proper folding; biasing G/Ccontent to increase mRNA stability or reduce secondary structures;minimizing tandem repeat codons or base runs that can impair geneconstruction or expression; customizing transcriptional andtranslational control regions; inserting or removing protein traffickingsequences; removing/adding post translation modification sites in anencoded protein (e.g., glycosylation sites); adding, removing orshuffling protein domains; inserting or deleting restriction sites;modifying ribosome binding sites and mRNA degradation sites; adjustingtranslational rates to allow the various domains of the protein to foldproperly; and/or reducing or eliminating problem secondary structureswithin the polynucleotide. Sequence optimization tools, algorithms andservices are known in the art, non-limiting examples include servicesfrom GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/orproprietary methods.

Codon options for each amino acid are given in TABLE 1.

TABLE 1 Codon Options Single Letter Amino Acid Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocysteine insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

In some embodiments, a polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a sequence-optimized nucleotide sequence (e.g.,an ORF) encoding a Citrin polypeptide, a functional fragment, or avariant thereof, wherein the Citrin polypeptide, functional fragment, ora variant thereof encoded by the sequence-optimized nucleotide sequencehas improved properties (e.g., compared to a Citrin polypeptide,functional fragment, or a variant thereof encoded by a referencenucleotide sequence that is not sequence optimized), e.g., improvedproperties related to expression efficacy after administration in vivo.Such properties include, but are not limited to, improving nucleic acidstability (e.g., mRNA stability), increasing translation efficacy in thetarget tissue, reducing the number of truncated proteins expressed,improving the folding or prevent misfolding of the expressed proteins,reducing toxicity of the expressed products, reducing cell death causedby the expressed products, increasing and/or decreasing proteinaggregation.

In some embodiments, the sequence-optimized nucleotide sequence is codonoptimized for expression in human subjects, having structural and/orchemical features that avoid one or more of the problems in the art, forexample, features which are useful for optimizing formulation anddelivery of nucleic acid-based therapeutics while retaining structuraland functional integrity; overcoming a threshold of expression;improving expression rates; half-life and/or protein concentrations;optimizing protein localization; and avoiding deleterious bio-responsessuch as the immune response and/or degradation pathways.

In some embodiments, the polynucleotides of the invention comprise anucleotide sequence (e.g., a nucleotide sequence (e.g., an ORF) encodinga Citrin polypeptide, a nucleotide sequence (e.g., an ORF) encodinganother polypeptide of interest, a 5′-UTR, a 3′-UTR, a microRNA bindingsite, a nucleic acid sequence encoding a linker, or any combinationthereof) that is sequence-optimized according to a method comprising:

-   -   (i) substituting at least one codon in a reference nucleotide        sequence (e.g., an ORF encoding a Citrin polypeptide) with an        alternative codon to increase or decrease uridine content to        generate a uridine-modified sequence;    -   (ii) substituting at least one codon in a reference nucleotide        sequence (e.g., an ORF encoding a Citrin polypeptide) with an        alternative codon having a higher codon frequency in the        synonymous codon set;    -   (iii) substituting at least one codon in a reference nucleotide        sequence (e.g., an ORF encoding a Citrin polypeptide) with an        alternative codon to increase G/C content; or    -   (iv) a combination thereof.

In some embodiments, the sequence-optimized nucleotide sequence (e.g.,an ORF encoding a Citrin polypeptide) has at least one improved propertywith respect to the reference nucleotide sequence.

In some embodiments, the sequence optimization method is multiparametricand comprises one, two, three, four, or more methods disclosed hereinand/or other optimization methods known in the art.

Features, which can be considered beneficial in some embodiments of theinvention, can be encoded by or within regions of the polynucleotide andsuch regions can be upstream (5′) to, downstream (3′) to, or within theregion that encodes the Citrin polypeptide. These regions can beincorporated into the polynucleotide before and/or aftersequence-optimization of the protein encoding region or open readingframe (ORF). Examples of such features include, but are not limited to,untranslated regions (UTRs), microRNA sequences, Kozak sequences,oligo(dT) sequences, poly-A tail, and detectable tags and can includemultiple cloning sites that can have XbaI recognition.

In some embodiments, the polynucleotide of the invention comprises a 5′UTR. a 3′ UTR and/or a miRNA binding site. In some embodiments, thepolynucleotide comprises two or more 5′ UTRs and/or 3′ UTRs, which canbe the same or different sequences. In some embodiments, thepolynucleotide comprises two or more miRNA, which can be the same ordifferent sequences. Any portion of the 5′ UTR, 3′ UTR, and/or miRNAbinding site, including none, can be sequence-optimized and canindependently contain one or more different structural or chemicalmodifications, before and/or after sequence optimization.

In some embodiments, after optimization, the polynucleotide isreconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized polynucleotide can be reconstituted and transformed intochemically competent E. coli, yeast, neurospora, maize, drosophila, etc.where high copy plasmid-like or chromosome structures occur by methodsdescribed herein.

6. Sequence-Optimized Nucleotide Sequences Encoding Citrin Polypeptides

In some embodiments, the polynucleotide of the invention comprises asequence-optimized nucleotide sequence encoding a Citrin polypeptidedisclosed herein. In some embodiments, the polynucleotide of theinvention comprises an open reading frame (ORF) encoding a Citrinpolypeptide, wherein the ORF has been sequence optimized.

Exemplary sequence-optimized nucleotide sequences encoding human Citrinisoform 1 are set forth as SEQ ID Nos: 5-29 (Citrin-CO01, Citrin-CO02,Citrin-CO03, Citrin-CO04, Citrin-COOS, Citrin-CO06, Citrin-CO07,Citrin-CO08, Citrin-CO09, Citrin-CO10, Citrin-CO11, Citrin-CO12,Citrin-CO13, Citrin-CO14, Citrin-CO15, Citrin-CO16, Citrin-CO17,Citrin-CO18, Citrin-CO19, Citrin-CO20, Citrin-CO21, Citrin-CO22,Citrin-CO23, Citrin-CO24, and Citrin-CO25, respectively. Furtherexemplary sequence optimized nucleotide sequences encoding human Citrinisoform 1 are shown in TABLE 2. In some embodiments, the sequenceoptimized Citrin sequences set forth as SEQ ID Nos: 5-29 or shown inTABLE 2, fragments, and variants thereof are used to practice themethods disclosed herein. In some embodiments, the sequence optimizedCitrin sequences set forth as SEQ ID Nos: 5-29 or shown in TABLE 2,fragments and variants thereof are combined with or alternatives to thewild-type sequences disclosed in FIGS. 1-2.

Exemplary sequence optimized nucleotide sequence encoding human Citrinisoform 1 are set forth as SEQ ID Nos: 129 (Construct #1 ORF), 132(Construct #2 ORF), 135 (Construct #3 ORF), and 138 (Construct #4 ORF).Further exemplary sequence optimized nucleotide sequences encoding humanCitrin isoform 1 are shown in TABLE 2. In some embodiments, the sequenceoptimized Citrin sequences set forth as SEQ ID Nos: 129, 132, 135, and138, or shown in TABLE 2, fragments, and variants thereof are used topractice the methods disclosed herein. In some embodiments, the sequenceoptimized Citrin sequences set forth as SEQ ID Nos: 129, 132, 135, and138, or shown in TABLE 2, fragments and variants thereof are combinedwith or alternatives to the wild-type sequences disclosed in FIGS. 1-2.

In some embodiments, a polynucleotide of the present disclosure, forexample a polynucleotide comprising an mRNA nucleotide sequence encodinga Citrin polypeptide, comprises from 5′ to 3′ end:

-   -   (i) a 5′ cap provided herein, for example, CAP1;    -   (ii) a 5′ UTR, such as the sequences provided herein, for        example, SEQ ID NO: 30;    -   (iii) an open reading frame encoding a Citrin polypeptide, e.g.,        a sequence optimized nucleic acid sequence encoding Citrin set        forth as SEQ ID Nos: 5 to 29, 129, 132, 135, and 138, or shown        in TABLE 2;    -   (iv) at least one stop codon;    -   (v) a 3′ UTR, such as the sequences provided herein, for        example, SEQ ID NO: 105, 147 and 148; and    -   (vi) a poly-A tail provided above.

TABLE 2 Sequence optimized sequences for human Citrin, isoform 1 SEQ IDNO Name Sequence   5 Citrin- See Sequence Listing CO01   6 Citrin-See Sequence Listing CO02   7 Citrin- See Sequence Listing CO03   8Citrin- See Sequence Listing CO04   9 Citrin- See Sequence Listing CO05 10 Citrin- See Sequence Listing CO06  11 Citrin- See Sequence ListingCO07  12 citrin- See Sequence Listing CO08  13 Citrin-See Sequence Listing CO09  14 citrin- See Sequence Listing CO10  15Citrin- See Sequence Listing CO11  16 citrin- See Sequence Listing CO12 17 Citrin- See Sequence Listing CO13  18 citrin- See Sequence ListingCO14  19 Citrin- See Sequence Listing CO15  20 Citrin-See Sequence Listing CO16  21 Citrin- See Sequence Listing CO17  22Citrin- See Sequence Listing CO18  23 Citrin- See Sequence Listing CO19 24 Citrin- See Sequence Listing CO20  25 Citrin- See Sequence ListingCO21  26 Citrin- See Sequence Listing CO22  27 Citrin-See Sequence Listing CO23  28 Citrin- See Sequence Listing CO24  29Citrin- See Sequence Listing CO25 129 ConstructAUGGCCGCCGCCAAGGUCGCCCUCACCAAGAGGGCGGACCCCGCGGAGCUCAGGACCAUCUUUCUCAAGU1 ORFACGCCUCCAUCGAGAAGAACGGGGAGUUCUUCAUGAGCCCGAACGAUUUCGUCACCAGGUACCUCAACAUCUUCGGCGAGAGCCAGCCCAACCCGAAGACCGUAGAGCUCCUCAGCGGCGUCGUCGACCAGACGAAGGACGGACUCAUUAGCUUCCAGGAGUUCGUCGCCUUCGAGAGCGUCCUCUGCGCGCCCGACGCCCUCUUCAUGGUCGCGUUCCAGCUGUUCGAUAAGGCCGGCAAGGGCGAGGUGACCUUUGAAGAUGUGAAGCAGGUGUUCGGUCAGACCACCAUCCACCAGCACAUCCCUUUUAACUGGGACUCAGAAUUCGUCCAGCUGCACUUCGGCAAGGAAAGGAAGCGCCAUCUGACCUACGCCGAGUUCACACAGUUUCUCCUGGAGAUCCAGCUGGAACACGCCAAACAGGCGUUCGUUCAGCGGGACAACGCCAGGACCGGCCGGGUCACCGCGAUCGACUUCCGUGAUAUCAUGGUGACCAUCAGGCCUCACGUCCUGACGCCCUUCGUGGAAGAGUGCCUGGUGGCCGCCGCGGGUGGGACCACGUCCCACCAGGUGAGCUUCUCCUAUUUUAACGGGUUCAACAGCCUCCUCAACAACAUGGAGCUGAUCCGGAAGAUCUAUAGCACCCUGGCCGGGACGCGCAAGGACGUCGAGGUGACCAAGGAAGAAUUCGUGCUGGCCGCCCAGAAGUUUGGCCAGGUGACGCCCAUGGAGGUGGACAUCCUGUUCCAACUGGCGGACCUGUACGAACCCAGGGGCCGGAUGACCCUGGCCGACAUCGAGCGCAUUGCCCCGCUCGAGGAGGGCACCCUGCCCUUCAACCUGGCGGAGGCCCAGCGCCAGAAGGCCAGCGGGGACAGCGCCAGGCCCGUCCUGCUGCAGGUGGCCGAGUCCGCGUACAGGUUCGGUCUGGGGAGCGUCGCGGGAGCCGUGGGUGCCACCGCCGUGUAUCCGAUCGACCUCGUGAAGACCCGUAUGCAGAAUCAGAGGUCCACCGGCAGCUUCGUGGGCGAGCUCAUGUACAAGAACUCCUUCGACUGUUUCAAGAAGGUUCUCCGGUACGAGGGGUUCUUCGGCCUCUACCGGGGACUGCUACCCCAGCUGCUCGGCGUGGCGCCCGAGAAGGCCAUCAAGCUCACGGUCAACGACUUUGUGCGGGACAAGUUCAUGCACAAGGACGGCAGCGUCCCACUGGCCGCCGAGAUUCUGGCAGGCGGCUGCGCCGGAGGCAGCCAGGUCAUCUUUACCAACCCACUGGAGAUCGUGAAGAUCAGGCUGCAAGUGGCCGGGGAGAUUACCACCGGACCCCGUGUGAGCGCCCUGAGCGUGGUGAGGGACCUUGGCUUCUUUGGCAUAUACAAGGGAGCCAAGGCCUGCUUUCUGAGGGACAUCCCGUUUUCCGCCAUAUACUUUCCCUGCUACGCCCACGUGAAAGCGAGCUUCGCCAACGAGGACGGCCAGGUCAGUCCCGGCUCCCUACUGCUGGCCGGAGCCAUCGCCGGGAUGCCCGCCGCCAGCCUGGUGACGCCCGCGGACGUUAUCAAGACCAGGCUGCAGGUGGCGGCCAGGGCGGGCCAGACCACCUACAGCGGCGUGAUCGACUGCUUCCGCAAGAUCCUGCGGGAGGAGGGGCCCAAGGCCCUGUGGAAAGGCGCCGGGGCAAGGGUGUUCAGGAGCAGCCCACAGUUCGGGGUGACCCUGCUUACCUACGAACUGCUGCAGCGGUGGUUCUAUAUCGACUUCGGAGGAGUCAAGCCCAUGGGCUCCGAGCCUGUGCCCAAGAGCAGGAUCAAUCUGCCCGCGCCCAACCCAGACCACGUGGGAGGGUACAAACUGGCCGUGGCCACCUUCGCCGGAAUCGAGAACAAGUUCGGCCUGUAUCUGCCCCUGUUCAAACCGUCCGUCUCCACCAGCAAGGCCAUCGGCGGAGGCCCC 132ConstructAUGGCCGCCGCCAAGGUCGCCCUCACCAAGAGGGCGGACCCCGCGGAGCUCAGGACCAUCUUUCUCAAGU2 ORFACGCCUCCAUCGAGAAGAACGGGGAGUUCUUCAUGAGCCCGAACGAUUUCGUCACCAGGUACCUCAACAUCUUCGGCGAGAGCCAGCCCAACCCGAAGACCGUAGAGCUCCUCAGCGGCGUCGUCGACCAGACGAAGGACGGACUCAUUAGCUUCCAGGAGUUCGUCGCCUUCGAGAGCGUCCUCUGCGCCCCCGACGCCCUCUUCAUGGUCGCGUUCCAGCUGUUCGAUAAGGCCGGCAAGGGCGAGGUGACCUUUGAAGAUGUGAAGCAGGUGUUCGGUCAGACCACCAUCCACCAGCACAUCCCUUUUAACUGGGACUCAGAAUUCGUCCAGCUGCACUUCGGCAAGGAAAGGAAGCGCCAUCUGACCUACGCCGAGUUCACACAGUUUCUCCUGGAGAUCCAGCUGGAACACGCCAAACAGGCGUUCGUUCAGCGGGACAACGCCAGGACCGGCCGGGUCACCGCGAUCGACUUCCGUGAUAUCAUGGUGACCAUCAGGCCUCACGUCCUGACCCCCUUCGUGGAAGAGUGCCUGGUGGCCGCCGCGGGUGGGACCACGUCCCACCAGGUGAGCUUCUCCUAUUUUAACGGGUUCAACAGCCUCCUCAACAACAUGGAGCUGAUCCGGAAGAUCUAUAGCACCCUGGCCGGGACGCGCAAGGACGUCGAGGUGACCAAGGAAGAAUUCGUGCUGGCCGCCCAGAAGUUUGGCCAGGUGACCCCCAUGGAGGUGGACAUCCUGUUCCAACUGGCGGACCUGUACGAACCCAGGGGCCGGAUGACCCUGGCCGACAUCGAGCGCAUUGCCCCGCUCGAGGAGGGCACCCUGCCCUUCAACCUGGCGGAGGCCCAGCGCCAGAAGGCCAGCGGGGACAGCGCCAGGCCCGUCCUGCUGCAGGUGGCCGAGUCCGCGUACAGGUUCGGUCUGGGGAGCGUCGCGGGAGCCGUGGGUGCCACCGCCGUGUAUCCGAUCGACCUCGUGAAGACCCGUAUGCAGAAUCAGAGGUCCACCGGCAGCUUCGUGGGCGAGCUCAUGUACAAGAACUCCUUCGACUGUUUCAAGAAGGUUCUCCGGUACGAGGGGUUCUUCGGCCUCUACCGGGGGCUGCUACCCCAGCUGCUCGGCGUGGCGCCCGAGAAGGCCAUCAAGCUCACGGUCAACGACUUUGUGCGGGACAAGUUCAUGCACAAGGACGGCAGCGUCCCCCUGGCCGCCGAGAUUCUGGCAGGCGGCUGCGCCGGAGGCAGCCAGGUCAUCUUUACCAACCCCCUGGAGAUCGUGAAGAUCAGGCUGCAAGUGGCCGGGGAGAUUACCACCGGACCCCGUGUGAGCGCCCUGAGCGUGGUGAGGGACCUUGGCUUCUUUGGCAUAUACAAGGGAGCCAAGGCCUGCUUUCUGAGGGACAUCCCGUUUUCCGCCAUAUACUUUCCCUGCUACGCCCACGUGAAAGCGAGCUUCGCCAACGAGGACGGCCAGGUCAGUCCCGGCUCCCUACUGCUGGCCGGAGCCAUCGCCGGGAUGCCCGCCGCCAGCCUGGUGACCCCCGCGGACGUUAUCAAGACCAGGCUGCAGGUGGCGGCCAGGGCGGGCCAGACCACCUACAGCGGCGUGAUCGACUGCUUCCGCAAGAUCCUGCGGGAGGAGGGGCCCAAGGCCCUGUGGAAAGGCGCCGGGGCAAGGGUGUUCAGGAGCAGCCCCCAGUUCGGGGUGACCCUGCUUACCUACGAACUGCUGCAGCGGUGGUUCUAUAUCGACUUCGGGGGAGUCAAGCCCAUGGGCUCCGAGCCUGUGCCCAAGAGCAGGAUCAAUCUGCCCGCCCCCAACCCAGACCACGUGGGAGGGUACAAACUGGCCGUGGCCACCUUCGCCGGAAUCGAGAACAAGUUCGGCCUGUAUCUGCCCCUGUUCAAACCGUCCGUCUCCACCAGCAAGGCCAUCGGCGGGGGCCCC 135ConstructAUGGCCGCCGCCAAGGUCGCACUCACCAAACGGGCCGACCCCGCCGAGCUCAGGACCAUCUUCCUCAAGU3 ORFACGCCAGCAUCGAGAAGAACGGGGAGUUCUUCAUGAGCCCCAACGACUUCGUAACCAGGUACCUCAAUAUCUUCGGCGAAAGCCAGCCCAACCCGAAGACCGUCGAGCUCCUCUCCGGGGUCGUCGAUCAGACCAAGGACGGGCUCAUCUCCUUCCAGGAGUUCGUGGCCUUCGAGUCCGUCCUCUGCGCCCCGGACGCCCUCUUCAUGGUCGCCUUCCAACUCUUCGAUAAGGCCGGGAAAGGCGAGGUGACCUUCGAAGAUGUGAAGCAGGUUUUCGGCCAGACCACGAUCCACCAGCAUAUCCCCUUUAACUGGGACAGCGAGUUCGUGCAGCUGCACUUCGGCAAAGAGAGGAAGAGGCACCUCACCUAUGCCGAAUUCACACAAUUCCUGCUCGAGAUUCAGCUGGAGCAUGCCAAACAAGCCUUUGUCCAGCGGGACAACGCCAGGACCGGCAGGGUCACCGCGAUCGACUUUCGCGAUAUCAUGGUGACCAUCAGGCCCCACGUGCUCACCCCCUUUGUGGAGGAGUGCCUGGUCGCCGCCGCCGGCGGGACGACCAGCCACCAGGUGUCCUUCUCAUACUUCAACGGGUUCAACAGCCUGCUGAACAACAUGGAGCUGAUCCGCAAGAUCUACUCCACCCUGGCCGGCACCAGGAAGGACGUGGAAGUGACCAAGGAGGAGUUCGUUCUCGCCGCCCAGAAAUUCGGACAGGUGACCCCGAUGGAGGUGGACAUCCUCUUCCAGCUGGCCGAUCUCUACGAGCCCCGGGGCAGGAUGACGCUCGCCGACAUCGAGCGAAUCGCCCCCCUGGAGGAGGGUACCCUUCCCUUCAACCUGGCCGAGGCCCAGCGACAGAAAGCCAGCGGCGACAGUGCCAGGCCCGUGCUCCUGCAGGUGGCCGAAUCCGCCUACAGAUUUGGCCUGGGCAGCGUCGCCGGCGCAGUGGGCGCCACCGCCGUGUACCCGAUCGACCUGGUCAAGACCAGGAUGCAGAACCAGAGAAGCACCGGCUCCUUUGUGGGCGAGCUUAUGUACAAGAACAGCUUCGACUGCUUCAAGAAGGUCCUGCGUUACGAGGGCUUCUUCGGGCUGUACCGGGGCCUGCUGCCGCAGCUGCUAGGCGUCGCGCCCGAGAAGGCCAUUAAGCUGACCGUGAACGACUUCGUACGGGACAAAUUCAUGCACAAGGACGGCAGUGUGCCCCUGGCCGCAGAGAUCCUGGCCGGGGGCUGUGCCGGCGGGUCCCAGGUGAUCUUUACGAACCCCCUGGAGAUAGUGAAGAUCCGGCUGCAGGUGGCCGGGGAGAUCACCACCGGCCCCCGGGUGUCCGCCCUGAGCGUCGUGAGGGAUCUGGGGUUCUUUGGCAUCUAUAAGGGUGCCAAGGCCUGCUUCCUGCGUGACAUUCCCUUUAGCGCCAUCUACUUCCCCUGCUACGCCCAUGUCAAGGCCAGCUUCGCCAACGAAGACGGACAGGUGAGCCCCGGGUCCCUGCUGCUGGCCGGCGCCAUCGCAGGGAUGCCGGCCGCCUCACUGGUGACCCCGGCGGACGUGAUAAAGACGAGGCUGCAGGUGGCCGCCCGCGCAGGCCAGACCACGUACUCGGGUGUGAUAGACUGCUUCCGGAAGAUCCUGAGGGAAGAAGGCCCCAAGGCCCUGUGGAAGGGGGCCGGCGCCAGGGUGUUCCGCAGCAGCCCCCAGUUCGGGGUCACCCUGCUCACGUACGAGCUCCUGCAGAGGUGGUUCUACAUCGACUUUGGCGGGGUCAAGCCCAUGGGCAGUGAGCCGGUGCCCAAGAGCCGGAUCAAUCUGCCCGCCCCCAACCCUGACCACGUCGGCGGGUACAAACUGGCGGUGGCCACCUUCGCCGGCAUCGAGAACAAGUUUGGCCUGUACCUGCCGCUGUUUAAGCCGUCCGUGAGCACCUCCAAGGCCAUCGGGGGCGGCCCA 138ConstructAUGGCCGCCGCUAAGGUCGCCCUCACGAAGCGGGCCGAUCCCGCCGAGCUCCGGACCAUCUUUCUCAAAU4 ORFACGCCAGCAUCGAGAAGAACGGCGAGUUCUUCAUGUCCCCCAACGACUUCGUCACCAGGUACCUAAACAUCUUCGGCGAGAGCCAGCCGAACCCCAAGACCGUCGAAUUGCUAUCGGGCGUCGUCGACCAGACCAAGGACGGGCUCAUCAGCUUCCAGGAGUUCGUCGCCUUCGAGUCCGUCCUCUGCGCCCCCGACGCCCUCUUCAUGGUAGCAUUCCAGUUGUUCGACAAGGCCGGAAAGGGGGAGGUGACCUUCGAGGACGUGAAGCAGGUGUUUGGUCAGACCACCAUCCAUCAGCACAUCCCGUUUAACUGGGACUCGGAGUUCGUGCAGCUCCACUUCGGCAAGGAACGAAAGCGGCACCUCACCUACGCCGAGUUCACCCAGUUCCUGCUGGAGAUCCAGCUGGAACACGCCAAGCAGGCCUUCGUGCAGCGAGACAAUGCGAGGACCGGGCGCGUGACUGCCAUCGAUUUCAGGGACAUCAUGGUGACCAUCCGGCCCCACGUGCUGACUCCCUUCGUGGAGGAGUGCCUGGUCGCAGCCGCCGGGGGUACCACCUCCCACCAGGUGAGCUUCAGCUACUUCAACGGCUUCAACAGCCUGCUCAACAACAUGGAACUGAUCCGCAAGAUCUAUUCCACACUCGCCGGCACCAGGAAGGAUGUGGAGGUGACCAAGGAGGAGUUUGUGCUGGCGGCCCAGAAGUUUGGGCAGGUCACCCCCAUGGAGGUGGACAUCCUCUUCCAGCUGGCCGACCUGUACGAGCCCAGGGGCAGGAUGACCCUGGCCGACAUCGAACGAAUCGCCCCCCUGGAGGAGGGCACACUGCCCUUCAACCUGGCCGAGGCCCAGAGGCAGAAGGCCUCCGGCGACAGCGCCCGACCCGUGCUUCUGCAAGUGGCCGAGAGCGCCUACCGCUUUGGCCUGGGAUCCGUGGCCGGCGCCGUCGGUGCCACCGCCGUGUACCCAAUCGACCUAGUGAAGACACGCAUGCAGAACCAGCGCAGCACCGGCUCCUUUGUCGGGGAGCUGAUGUAUAAGAACAGCUUCGACUGCUUCAAGAAGGUGCUGCGCUACGAGGGCUUCUUCGGCCUGUACAGGGGGCUGCUGCCCCAACUGCUGGGCGUGGCCCCCGAGAAGGCCAUCAAGCUGACCGUUAACGACUUCGUGCGAGACAAGUUCAUGCACAAGGACGGCAGCGUGCCCUUGGCCGCAGAGAUACUGGCCGGGGGCUGCGCCGGCGGGUCGCAGGUGAUCUUCACCAACCCCCUCGAGAUCGUAAAGAUCAGGCUCCAAGUGGCCGGGGAGAUCACCACCGGCCCCAGGGUGUCCGCCCUGUCGGUGGUCAGGGACCUGGGGUUCUUCGGGAUAUACAAGGGCGCGAAGGCCUGCUUCCUGAGGGACAUCCCCUUCAGCGCCAUCUACUUUCCCUGCUACGCCCACGUCAAGGCCAGCUUCGCCAACGAGGAUGGCCAGGUGUCCCCGGGCUCCCUGCUCCUGGCCGGUGCUAUCGCGGGAAUGCCCGCCGCGAGCCUGGUGACCCCCGCCGAUGUGAUCAAGACCAGGCUGCAAGUCGCGGCGAGGGCCGGUCAGACCACCUACUCCGGGGUCAUCGAUUGCUUUAGGAAGAUCCUCCGGGAGGAGGGGCCCAAGGCACUGUGGAAGGGGGCCGGGGCCAGGGUGUUCCGUAGCUCCCCCCAGUUUGGCGUGACCCUGCUGACCUACGAACUGCUGCAGAGGUGGUUCUACAUCGACUUCGGGGGCGUGAAGCCCAUGGGCUCCGAGCCGGUGCCUAAGAGCCGGAUCAAUCUGCCCGCCOCCAACCCCGACCACGUGGGGGGCUACAAGOUGGCCGUGGCUACCUUCGCCGGUAUCGAGAACAAGUUCGGCCUGUACCUGOCCCUGUUCAAGCCCUCCGUGAGCACGAGCAAGGCCAUCGGGGGCGGCCCC

The sequence-optimized nucleotide sequences disclosed herein aredistinct from the corresponding wild type nucleotide acid sequences andfrom other known sequence-optimized nucleotide sequences, e.g., thesesequence-optimized nucleic acids have unique compositionalcharacteristics.

In some embodiments, the percentage of uracil or thymine nucleobases ina sequence-optimized nucleotide sequence (e.g., encoding a Citrinpolypeptide, a functional fragment, or a variant thereof) is modified(e.g., reduced) with respect to the percentage of uracil or thyminenucleobases in the reference wild-type nucleotide sequence. Such asequence is referred to as a uracil-modified or thymine-modifiedsequence. The percentage of uracil or thymine content in a nucleotidesequence can be determined by dividing the number of uracils or thyminesin a sequence by the total number of nucleotides and multiplying by 100.In some embodiments, the sequence-optimized nucleotide sequence has alower uracil or thymine content than the uracil or thymine content inthe reference wild-type sequence. In some embodiments, the uracil orthymine content in a sequence-optimized nucleotide sequence of theinvention is greater than the uracil or thymine content in the referencewild-type sequence and still maintain beneficial effects, e.g.,increased expression and/or reduced Toll-Like Receptor (TLR) responsewhen compared to the reference wild-type sequence.

The uracil or thymine content of wild-type Citrin isoform 1 is about28%. In some embodiments, the uracil or thymine content of a uracil- orthymine-modified sequence encoding a Citrin polypeptide is less than28%. In some embodiments, the uracil or thymine content of a uracil- orthymine-modified sequence encoding a Citrin polypeptide of the inventionis less than 27%, less than 26%, less than 25%, less than 24%, less than23%, less than 22%, less than 21%, less than 20%, less than 19%, lessthat 18%, less than 17%, less than 16%, less than 15%, less than 14%,less than 13%, less than 12%, less than 11%, or less than 10%. In someembodiments, the uracil or thymine content is not less than 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, or 10%. The uracil or thymine content of asequence disclosed herein, i.e., its total uracil or thymine content isabbreviated herein as % Um or % Tm.

In some embodiments, the uracil or thymine content (% U_(TL), or %T_(TL)) of a uracil- or thymine-modified sequence encoding a Citrinpolypeptide of the invention is between 15% and 20%, between 16% and20%, between 16% and 19%, between 17% and 19%, or between 17% and 18%.

In some embodiments, the uracil or thymine content (% U_(TL), or %T_(TL)) of a uracil- or thymine-modified sequence encoding a Citrinpolypeptide of the invention is between 15% and 20%, between 15% and19%, between 16% and 19%, or between 15% and 18%.

In a particular embodiment, the uracil or thymine content (% U_(TL), or% T_(TL)) of a uracil- or thymine modified sequence encoding a Citrinpolypeptide of the invention is between about 16% and about 18%.

A uracil- or thymine-modified sequence encoding a Citrin polypeptide ofthe invention can also be described according to its uracil or thyminecontent relative to the uracil or thymine content in the correspondingwild-type nucleic acid sequence (% U_(WT) or % T_(WT)), or according toits uracil or thymine content relative to the theoretical minimum uracilor thymine content of a nucleic acid encoding the wild-type proteinsequence (% U_(TM) or (% T_(TM)).

The phrases “uracil or thymine content relative to the uracil or thyminecontent in the wild type nucleic acid sequence,” refers to a parameterdetermined by dividing the number of uracils or thymines in asequence-optimized nucleic acid by the total number of uracils orthymines in the corresponding wild-type nucleic acid sequence andmultiplying by 100. This parameter is abbreviated herein as % U_(WT) or% T_(WT).

In some embodiments, the % U_(WT) or % T_(WT) of a uracil- orthymine-modified sequence encoding a Citrin polypeptide of the inventionis above 50%, above 55%, above 60%, above 65%, above 70%, above 75%,above 80%, above 85%, above 90%, or above 95%.

In some embodiments, the % U_(WT) or % T_(WT) of a uracil- or thyminemodified sequence encoding a Citrin polypeptide of the invention isbetween 50% and 85%, between 51% and 84%, between 52% and 83%, between53% and 82%, between 54% and 81%, between 55% and 80%, between 56% and79%, between 57% and 78%, between 58% and 77%, between 59% and 76%,between 60% and 75%, or between 60% and 74%.

In some embodiments, the % U_(WT) or % T_(WT) of a uracil- orthymine-modified sequence encoding a Citrin polypeptide of the inventionis between 58% and 67%, between 59% and 66%, between 59% and 65%, orbetween 60% and 65%.

In a particular embodiment, the % U_(WT) or % T_(WT) of a uracil- orthymine-modified sequence encoding a Citrin polypeptide of the inventionis between about 60% and about 65%.

Uracil- or thymine-content relative to the uracil or thymine theoreticalminimum, refers to a parameter determined by dividing the number ofuracils or thymines in a sequence-optimized nucleotide sequence by thetotal number of uracils or thymines in a hypothetical nucleotidesequence in which all the codons in the hypothetical sequence arereplaced with synonymous codons having the lowest possible uracil orthymine content and multiplying by 100. This parameter is abbreviatedherein as % U_(TM) or % T_(TM).

For DNA it is recognized that thymine is present instead of uracil, andone would substitute T where U appears. Thus, all the disclosuresrelated to, e.g., % U_(TM), % U_(WT), or % U_(TL), with respect to RNAare equally applicable to % T_(TM), % T_(WT), or % T_(TL) with respectto DNA.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodinga Citrin polypeptide of the invention is below 300%, below 295%, below290%, below 285%, below 280%, below 275%, below 270%, below 265%, below260%, below 255%, below 250%, below 245%, below 240%, below 235%, below230%, below 225%, below 220%, below 215%, below 200%, below 195%, below190%, below 185%, below 180%, below 175%, below 170%, below 165%, below160%, below 155%, below 150%, below 145%, below 140%, below 139%, below138%, below 137%, below 136%, below 135%, below 134%, below 133%, below132%, below 131%, below 130%, below 129%, below 128%, below 127%, below126%, below 125%, below 124%, below 123%, below 122%, below 121%, below120%, below 119%, below 118%, below 117%, below 116%, or below 115%.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodinga Citrin polypeptide of the invention is above 100%, above 101%, above102%, above 103%, above 104%, above 105%, above 106%, above 107%, above108%, above 109%, above 110%, above 111%, above 112%, above 113%, above114%, above 115%, above 116%, above 117%, above 118%, above 119%, above120%, above 121%, above 122%, above 123%, above 124%, above 125%, orabove 126%.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodinga Citrin polypeptide of the invention is between 118% and 122%, between117% and 123%, between 116% and 124%, between 115% and 125%, between114% and 126%, between 113% and 127%, between 112% and 128%, between111% and 129%, between 110% and 130%, between 109% and 131%, between108% and 132%, between 107% and 133%, or between 106% and 134%.

In some embodiments, the % U_(TM) of a uracil-modified sequence encodinga Citrin polypeptide of the invention is between about 117% and about127%.

In some embodiments, a uracil-modified sequence encoding a Citrinpolypeptide of the invention has a reduced number of consecutive uracilswith respect to the corresponding wild-type nucleic acid sequence. Forexample, two consecutive leucines can be encoded by the sequence CUUUUG,which includes a four uracil cluster. Such a subsequence can besubstituted, e.g., with CUGCUC, which removes the uracil cluster.

Phenylalanine can be encoded by UUC or UUU. Thus, even if phenylalaninesencoded by UUU are replaced by UUC, the synonymous codon still containsa uracil pair (UU). Accordingly, the number of phenylalanines in asequence establishes a minimum number of uracil pairs (UU) that cannotbe eliminated without altering the number of phenylalanines in theencoded polypeptide. For example, if the polypeptide, e.g., wild typeCitrin isoform 1, has, e.g., 49, 50, 51, 52, or 53 phenylalanines, theabsolute minimum number of uracil pairs (UU) that a uracil-modifiedsequence encoding the polypeptide, e.g., wild type Citrin isoform 1, cancontain is 49, 50, 51, 52, or 53, respectively.

Wild type Citrin isoform 1 contains 61 uracil pairs (UU), and 37 uraciltriplets (UUU). In some embodiments, a uracil-modified sequence encodinga Citrin polypeptide of the invention has a reduced number of uraciltriplets (UUU) with respect to the wild-type nucleic acid sequence. Insome embodiments, a uracil-modified sequence encoding a Citrinpolypeptide of the invention contains 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, 1 or no uracil triplets (UUU).

In some embodiments, a uracil-modified sequence encoding a Citrinpolypeptide has a reduced number of uracil pairs (UU) with respect tothe number of uracil pairs (UU) in the wild-type nucleic acid sequence.In some embodiments, a uracil-modified sequence encoding a Citrinpolypeptide of the invention has a number of uracil pairs (UU)corresponding to the minimum possible number of uracil pairs (UU) in thewild-type nucleic acid sequence, e.g., 9 uracil pairs in the case ofwild type Citrin isoform 1.

In some embodiments, a uracil-modified sequence encoding a Citrinpolypeptide of the invention has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40or 45 uracil pairs (UU) less than the number of uracil pairs (UU) in thewild-type nucleic acid sequence. In some embodiments, a uracil-modifiedsequence encoding a Citrin polypeptide of the invention has between 30and 55 uracil pairs (UU), e.g., 34 and 52.

The phrase “uracil pairs (UU) relative to the uracil pairs (UU) in thewild type nucleic acid sequence,” refers to a parameter determined bydividing the number of uracil pairs (UU) in a sequence-optimizednucleotide sequence by the total number of uracil pairs (UU) in thecorresponding wild-type nucleotide sequence and multiplying by 100. Thisparameter is abbreviated herein as % UU_(wt).

In some embodiments, a uracil-modified sequence encoding a Citrinpolypeptide of the invention has a % UU_(wt) less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 65%, less than 60%, less than 55%, less than 50%,less than 40%, less than 30%, or less than 20%.

In some embodiments, a uracil-modified sequence encoding a Citrinpolypeptide has a % UU_(wt) between 50% and 90%. In a particularembodiment, a uracil-modified sequence encoding a Citrin polypeptide ofthe invention has a % UU_(wt) between 55% and 86%.

In some embodiments, the polynucleotide of the invention comprises auracil-modified sequence encoding a Citrin polypeptide disclosed herein.In some embodiments, the uracil-modified sequence encoding a Citrinpolypeptide comprises at least one chemically modified nucleobase, e.g.,5-methoxyuracil. In some embodiments, at least 95% of a nucleobase(e.g., uracil) in a uracil-modified sequence encoding a Citrinpolypeptide of the invention are modified nucleobases. In someembodiments, at least 95% of uracil in a uracil-modified sequenceencoding a Citrin polypeptide is 5-methoxyuracil. In some embodiments,the polynucleotide comprising a uracil-modified sequence furthercomprises a miRNA binding site, e.g., a miRNA binding site that binds tomiR-142 and/or a miRNA binding site that binds to miR-126. In someembodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprisinga uracil-modified sequence disclosed herein is formulated with adelivery agent comprising, e.g., a compound having the Formula (I),e.g., any of Compounds 1-232, e.g., Compound 18; a compound having theFormula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g.,Compound 236; or a compound having the Formula (VIII), e.g., any ofCompounds 419-428, e.g., Compound 428, or any combination thereof. Insome embodiments, the delivery agent comprises Compound 18, DSPC,Cholesterol, and Compound 428, e.g., with a mole ratio of about50:10:38.5:1.5.

In some embodiments, the “guanine content of the sequence optimized ORFencoding Citrin with respect to the theoretical maximum guanine contentof a nucleotide sequence encoding the Citrin polypeptide,” abbreviatedas % F_(TMX) is at least 59%, at least 60%, least 65, at least 69%, atleast 70%, at least 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%. In some embodiments,the % G_(TMX) is between about 70% and about 80%, between about 70% andabout 79%, between about 70% and about 78%, or between about 70% andabout 76%.

In some embodiments, the “cytosine content of the ORF relative to thetheoretical maximum cytosine content of a nucleotide sequence encodingthe Citrin polypeptide,” abbreviated as % C_(TMX), is at least 49%, atleast 50%, at least 55%, at least 59%, at least 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%. Insome embodiments, the % C_(TMX) is between about 60% and about 80%,between about 65% and about 80%, between about 65% and about 78%, orbetween about 69% and about 75%.

In some embodiments, the “guanine and cytosine content (G/C) of the ORFrelative to the theoretical maximum G/C content in a nucleotide sequenceencoding the Citrin polypeptide,” abbreviated as % G/C_(TMX) is at leastabout 68%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or about100%. The % G/C_(TMX) is between about 80% and about 100%, between about85% and about 98%, between about 90% and about 95%, or between about 91%and about 94%.

In some embodiments, the “G/C content in the ORF relative to the G/Ccontent in the corresponding wild-type ORF,” abbreviated as % G/CWT isat least 102%, at least 103%, at least 104%, at least 105%, at least106%, at least 107%, at least 110%, at least 115%, at least 120%, atleast 125%, at least 130%, or at least 135%.

In some embodiments, the average G/C content in the 3rd codon positionin the ORF is at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 35%, at least 40%, or at least 45%higher than the average G/C content in the 3rd codon position in thecorresponding wild-type ORF.

In some embodiments, the polynucleotide of the invention comprises anopen reading frame (ORF) encoding a Citrin polypeptide, wherein the ORFhas been sequence optimized, and wherein each of % U_(TL), % U_(WT), %U_(TM), % Gm, % G_(WT), % G_(TMX), % C_(TL), % C_(WT), % G_(TMX), %G/C_(TL), % G/C_(WT), or % G/C_(TMX), alone or in a combination thereofis in a range between (i) a maximum corresponding to the parameter'smaximum value (MAX) plus about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 standard deviations (STDDEV), and (ii) a minimum corresponding to the parameter's minimum value(MIN) less 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, or 10 standard deviations (STD DEV).

7. Methods for Sequence Optimization

In some embodiments, a polynucleotide, e.g., mRNA, of the invention(e.g., a polynucleotide comprising a nucleotide sequence encoding aCitrin polypeptide (e.g., the wild-type sequence, functional fragment,or variant thereof) is sequence optimized. A sequence optimizednucleotide sequence (nucleotide sequence is also referred to as “nucleicacid” herein) comprises at least one codon modification with respect toa reference sequence (e.g., a wild-type sequence encoding a Citrinpolypeptide). Thus, in a sequence optimized nucleic acid, at least onecodon is different from a corresponding codon in a reference sequence(e.g., a wild-type sequence).

In general, sequence optimized nucleic acids are generated by at least astep comprising substituting codons in a reference sequence withsynonymous codons (i.e., codons that encode the same amino acid). Suchsubstitutions can be effected, for example, by applying a codonsubstitution map (i.e., a table providing the codons that will encodeeach amino acid in the codon optimized sequence), or by applying a setof rules (e.g., if glycine is next to neutral amino acid, glycine wouldbe encoded by a certain codon, but if it is next to a polar amino acid,it would be encoded by another codon). In addition to codonsubstitutions (i.e., “codon optimization”) the sequence optimizationmethods disclosed herein comprise additional optimization steps whichare not strictly directed to codon optimization such as the removal ofdeleterious motifs (destabilizing motif substitution). Compositions andformulations comprising these sequence optimized nucleic acids (e.g., aRNA, e.g., an mRNA) can be administered to a subject in need thereof tofacilitate in vivo expression of functionally active Citrin.

The recombinant expression of large molecules in cell cultures can be achallenging task with numerous limitations (e.g., poor proteinexpression levels, stalled translation resulting in truncated expressionproducts, protein misfolding, etc.) These limitations can be reduced oravoided by administering the polynucleotides (e.g., a RNA, e.g., anmRNA), which encode a functionally active Citrin or compositions orformulations comprising the same to a patient suffering fromCitrullinemia Type 2 (CTLN2), so the synthesis and delivery of theCitrin polypeptide to treat CTLN2 takes place endogenously.

Changing from an in vitro expression system (e.g., cell culture) to invivo expression requires the redesign of the nucleic acid sequenceencoding the therapeutic agent. Redesigning a naturally occurring genesequence by choosing different codons without necessarily altering theencoded amino acid sequence can often lead to dramatic increases inprotein expression levels (Gustafsson et al., 2004, Journal/TrendsBiotechnol 22, 346-53). Variables such as codon adaptation index (CAI),mRNA secondary structures, cis-regulatory sequences, GC content and manyother similar variables have been shown to somewhat correlate withprotein expression levels (Villalobos et al., 2006, “Journal/BMCBioinformatics 7, 285). However, due to the degeneracy of the geneticcode, there are numerous different nucleic acid sequences that can allencode the same therapeutic agent. Each amino acid is encoded by up tosix synonymous codons; and the choice between these codons influencesgene expression. In addition, codon usage (i.e., the frequency withwhich different organisms use codons for expressing a polypeptidesequence) differs among organisms (for example, recombinant productionof human or humanized therapeutic antibodies frequently takes place inhamster cell cultures).

In some embodiments, a reference nucleic acid sequence can be sequenceoptimized by applying a codon map. The skilled artisan will appreciatethat the T bases in the codon maps disclosed below are present in DNA,whereas the T bases would be replaced by U bases in corresponding RNAs.For example, a sequence optimized nucleic acid disclosed herein in DNAform, e.g., a vector or an in-vitro translation (IVT) template, wouldhave its T bases transcribed as U based in its corresponding transcribedmRNA. In this respect, both sequence optimized DNA sequences (comprisingT) and their corresponding RNA sequences (comprising U) are consideredsequence optimized nucleic acid of the present invention. A skilledartisan would also understand that equivalent codon-maps can begenerated by replaced one or more bases with non-natural bases. Thus,e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map),which in turn can correspond to a ΨΨC codon (RNA map in which U has beenreplaced with pseudouridine).

In one embodiment, a reference sequence encoding Citrin can be optimizedby replacing all the codons encoding a certain amino acid with only oneof the alternative codons provided in a codon map. For example, all thevalines in the optimized sequence would be encoded by GTG or GTC or GTT.

Sequence optimized polynucleotides of the invention can be generatedusing one or more codon optimization methods, or a combination thereof.Sequence optimization methods which can be used to sequence optimizenucleic acid sequences are described in detail herein. This list ofmethods is not comprehensive or limiting.

It will be appreciated that the design principles and rules describedfor each one of the sequence optimization methods discussed below can becombined in many different ways, for example high G/C content sequenceoptimization for some regions or uridine content sequence optimizationfor other regions of the reference nucleic acid sequence, as well astargeted nucleotide mutations to minimize secondary structure throughoutthe sequence or to eliminate deleterious motifs.

The choice of potential combinations of sequence optimization methodscan be, for example, dependent on the specific chemistry used to producea synthetic polynucleotide. Such a choice can also depend oncharacteristics of the protein encoded by the sequence optimized nucleicacid, e.g., a full sequence, a functional fragment, or a fusion proteincomprising Citrin, etc. In some embodiments, such a choice can depend onthe specific tissue or cell targeted by the sequence optimized nucleicacid (e.g., a therapeutic synthetic mRNA).

The mechanisms of combining the sequence optimization methods or designrules derived from the application and analysis of the optimizationmethods can be either simple or complex. For example, the combinationcan be:

-   -   (i) Sequential: Each sequence optimization method or set of        design rules applies to a different subsequence of the overall        sequence, for example reducing uridine at codon positions 1 to        30 and then selecting high frequency codons for the remainder of        the sequence;    -   (ii) Hierarchical: Several sequence optimization methods or sets        of design rules are combined in a hierarchical, deterministic        fashion. For example, use the most GC-rich codons, breaking ties        (which are common) by choosing the most frequent of those        codons.    -   (iii) Multifactorial/Multiparametric: Machine learning or other        modeling techniques are used to design a single sequence that        best satisfies multiple overlapping and possibly contradictory        requirements. This approach would require the use of a computer        applying a number of mathematical techniques, for example,        genetic algorithms.

Ultimately, each one of these approaches can result in a specific set ofrules which in many cases can be summarized in a single codon table,i.e., a sorted list of codons for each amino acid in the target protein(i.e., Citrin), with a specific rule or set of rules indicating how toselect a specific codon for each amino acid position.

a. Uridine Content Optimization

The presence of local high concentrations of uridine in a nucleic acidsequence can have detrimental effects on translation, e.g., slow orprematurely terminated translation, especially when modified uridineanalogs are used in the production of synthetic mRNAs. Furthermore, highuridine content can also reduce the in vivo half-life of synthetic mRNAsdue to TLR activation.

Accordingly, a nucleic acid sequence can be sequence optimized using amethod comprising at least one uridine content optimization step. Such astep comprises, e.g., substituting at least one codon in the referencenucleic acid with an alternative codon to generate a uridine-modifiedsequence, wherein the uridine-modified sequence has at least one of thefollowing properties:

-   -   (i) increase or decrease in global uridine content;    -   (ii) increase or decrease in local uridine content (i.e.,        changes in uridine content are limited to specific        subsequences);    -   (iii) changes in uridine distribution without altering the        global uridine content;    -   (iv) changes in uridine clustering (e.g., number of clusters,        location of clusters, or distance between clusters); or    -   (v) combinations thereof.

In some embodiments, the sequence optimization process comprisesoptimizing the global uridine content, i.e., optimizing the percentageof uridine nucleobases in the sequence optimized nucleic acid withrespect to the percentage of uridine nucleobases in the referencenucleic acid sequence. For example, 30% of nucleobases can be uridinesin the reference sequence and 10% of nucleobases can be uridines in thesequence optimized nucleic acid.

In other embodiments, the sequence optimization process comprisesreducing the local uridine content in specific regions of a referencenucleic acid sequence, i.e., reducing the percentage of uridinenucleobases in a subsequence of the sequence optimized nucleic acid withrespect to the percentage of uridine nucleobases in the correspondingsubsequence of the reference nucleic acid sequence. For example, thereference nucleic acid sequence can have a 5′-end region (e.g., 30codons) with a local uridine content of 30%, and the uridine content inthat same region could be reduced to 10% in the sequence optimizednucleic acid.

In specific embodiments, codons can be replaced in the reference nucleicacid sequence to reduce or modify, for example, the number, size,location, or distribution of uridine clusters that could havedeleterious effects on protein translation. Although as a general ruleit is desirable to reduce the uridine content of the reference nucleicacid sequence, in certain embodiments the uridine content, and inparticular the local uridine content, of some subsequences of thereference nucleic acid sequence can be increased.

The reduction of uridine content to avoid adverse effects on translationcan be done in combination with other optimization methods disclosedhere to achieve other design goals. For example, uridine contentoptimization can be combined with ramp design, since using the rarestcodons for most amino acids will, with a few exceptions, reduce the Ucontent.

In some embodiments, the uridine-modified sequence is designed to inducea lower Toll-Like Receptor (TLR) response when compared to the referencenucleic acid sequence. Several TLRs recognize and respond to nucleicacids. Double-stranded (ds)RNA, a frequent viral constituent, has beenshown to activate TLR3. See Alexopoulou et al. (2001) Nature,413:732-738 and Wang et al. (2004) Nat. Med., 10:1366-1373.Single-stranded (ss)RNA activates TLR7. See Diebold et al. (2004)Science 303:1529-1531. RNA oligonucleotides, for example RNA withphosphorothioate internucleotide linkages, are ligands of human TLR8.See Heil et al. (2004) Science 303:1526-1529. DNA containingunmethylated CpG motifs, characteristic of bacterial and viral DNA,activate TLR9. See Hemmi et al. (2000) Nature, 408: 740-745.

As used herein, the term “TLR response” is defined as the recognition ofsingle-stranded RNA by a TLR7 receptor, and in some embodimentsencompasses the degradation of the RNA and/or physiological responsescaused by the recognition of the single-stranded RNA by the receptor.Methods to determine and quantitate the binding of an RNA to a TLR7 areknown in the art. Similarly, methods to determine whether an RNA hastriggered a TLR7-mediated physiological response (e.g., cytokinesecretion) are well known in the art. In some embodiments, a TLRresponse can be mediated by TLR3, TLR8, or TLR9 instead of TLR7.

Suppression of TLR7-mediated response can be accomplished via nucleosidemodification. RNA undergoes over hundred different nucleosidemodifications in nature (see the RNA Modification Database, available atmods.ma.albany.edu). Human rRNA, for example, has ten times morepseudouridine (Ψ) and 25 times more 2′-O-methylated nucleosides thanbacterial rRNA. Bacterial mRNA contains no nucleoside modifications,whereas mammalian mRNAs have modified nucleosides such as5-methylcytidine (m5C), N6-methyladenosine (m6A), inosine and many2′-O-methylated nucleosides in addition to N7-methylguanosine (m7G).

Uracil and ribose, the two defining features of RNA, are both necessaryand sufficient for TLR7 stimulation, and short single-stranded RNA(ssRNA) act as TLR7 agonists in a sequence-independent manner as long asthey contain several uridines in close proximity. See Diebold et al.(2006) Eur. J. Immunol. 36:3256-3267, which is herein incorporated byreference in its entirety. Accordingly, one or more of the optimizationmethods disclosed herein comprises reducing the uridine content (locallyand/or locally) and/or reducing or modifying uridine clustering toreduce or to suppress a TLR7-mediated response.

In some embodiments, the TLR response (e.g., a response mediated byTLR7) caused by the uridine-modified sequence is at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or at leastabout 100% lower than the TLR response caused by the reference nucleicacid sequence.

In some embodiments, the TLR response caused by the reference nucleicacid sequence is at least about 1-fold, at least about 1.1-fold, atleast about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold,at least about 1.5-fold, at least about 1.6-fold, at least about1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at leastabout 2-fold, at least about 3-fold, at least about 4-fold, at leastabout 5-fold, at least about 6-fold, at least about 7-fold, at leastabout 8-fold, at least about 9-fold, or at least about 10-fold higherthan the TLR response caused by the uridine-modified sequence.

In some embodiments, the uridine content (average global uridinecontent) (absolute or relative) of the uridine-modified sequence ishigher than the uridine content (absolute or relative) of the referencenucleic acid sequence. Accordingly, in some embodiments, theuridine-modified sequence contains at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, or atleast about 100% more uridine that the reference nucleic acid sequence.

In other embodiments, the uridine content (average global uridinecontent) (absolute or relative) of the uridine-modified sequence islower than the uridine content (absolute or relative) of the referencenucleic acid sequence. Accordingly, in some embodiments, theuridine-modified sequence contains at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, or atleast about 100% less uridine that the reference nucleic acid sequence.

In some embodiments, the uridine content (average global uridinecontent) (absolute or relative) of the uridine-modified sequence is lessthan 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%,37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%,23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total nucleobases in theuridine-modified sequence. In some embodiments, the uridine content ofthe uridine-modified sequence is between about 10% and about 20%. Insome particular embodiments, the uridine content of the uridine-modifiedsequence is between about 12% and about 16%.

In some embodiments, the uridine content of the reference nucleic acidsequence can be measured using a sliding window. In some embodiments,the length of the sliding window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 nucleobases. In some embodiments, thesliding window is over 40 nucleobases in length. In some embodiments,the sliding window is 20 nucleobases in length. Based on the uridinecontent measured with a sliding window, it is possible to generate ahistogram representing the uridine content throughout the length of thereference nucleic acid sequence and sequence optimized nucleic acids.

In some embodiments, a reference nucleic acid sequence can be modifiedto reduce or eliminate peaks in the histogram that are above or below acertain percentage value. In some embodiments, the reference nucleicacid sequence can be modified to eliminate peaks in the sliding-windowrepresentation which are above 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30%uridine. In another embodiment, the reference nucleic acid sequence canbe modified so no peaks are over 30% uridine in the sequence optimizednucleic acid, as measured using a 20 nucleobase sliding window. In someembodiments, the reference nucleic acid sequence can be modified so nomore or no less than a predetermined number of peaks in the sequenceoptimized nucleic sequence, as measured using a 20 nucleobase slidingwindow, are above or below a certain threshold value. For example, insome embodiments, the reference nucleic acid sequence can be modified sono peaks or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peaks in thesequence optimized nucleic acid are above 10%, 15%, 20%, 25% or 30%uridine. In another embodiment, the sequence optimized nucleic acidcontains between 0 peaks and 2 peaks with uridine contents 30% ofhigher.

In some embodiments, a reference nucleic acid sequence can be sequenceoptimized to reduce the incidence of consecutive uridines. For example,two consecutive leucines could be encoded by the sequence CUUUUG, whichwould include a four uridine cluster. Such subsequence could besubstituted with CUGCUC, which would effectively remove the uridinecluster. Accordingly, a reference nucleic sequence can be sequenceoptimized by reducing or eliminating uridine pairs (UU), uridinetriplets (UUU) or uridine quadruplets (UUUU). Higher order combinationsof U are not considered combinations of lower order combinations. Thus,for example, UUUU is strictly considered a quadruplet, not twoconsecutive U pairs; or UUUUUU is considered a sextuplet, not threeconsecutive U pairs, or two consecutive U triplets, etc.

In some embodiments, all uridine pairs (UU) and/or uridine triplets(UUU) and/or uridine quadruplets (UUUU) can be removed from thereference nucleic acid sequence. In other embodiments, uridine pairs(UU) and/or uridine triplets (UUU) and/or uridine quadruplets (UUUU) canbe reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences inthe sequence optimized nucleic acid. In a particular embodiment, thesequence optimized nucleic acid contains less than 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 uridine pairs. Inanother particular embodiment, the sequence optimized nucleic acidcontains no uridine pairs and/or triplets.

Phenylalanine codons, i.e., UUC or UUU, comprise a uridine pair ortriples and therefore sequence optimization to reduce uridine contentcan at most reduce the phenylalanine U triplet to a phenylalanine Upair. In some embodiments, the occurrence of uridine pairs (UU) and/oruridine triplets (UUU) refers only to non-phenylalanine U pairs ortriplets. Accordingly, in some embodiments, non-phenylalanine uridinepairs (UU) and/or uridine triplets (UUU) can be reduced below a certainthreshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 occurrences in the sequence optimizednucleic acid. In a particular embodiment, the sequence optimized nucleicacid contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 non-phenylalanine uridine pairs and/ortriplets. In another particular embodiment, the sequence optimizednucleic acid contains no non-phenylalanine uridine pairs and/ortriplets.

In some embodiments, the reduction in uridine combinations (e.g., pairs,triplets, quadruplets) in the sequence optimized nucleic acid can beexpressed as a percentage reduction with respect to the uridinecombinations present in the reference nucleic acid sequence.

In some embodiments, a sequence optimized nucleic acid can contain about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ofthe total number of uridine pairs present in the reference nucleic acidsequence. In some embodiments, a sequence optimized nucleic acid cancontain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, or 65% of the total number of uridine triplets present in thereference nucleic acid sequence. In some embodiments, a sequenceoptimized nucleic acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, or 65% of the total number of uridinequadruplets present in the reference nucleic acid sequence.

In some embodiments, a sequence optimized nucleic acid can contain about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ofthe total number of non-phenylalanine uridine pairs present in thereference nucleic acid sequence. In some embodiments, a sequenceoptimized nucleic acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, or 65% of the total number ofnon-phenylalanine uridine triplets present in the reference nucleic acidsequence.

In some embodiments, the uridine content in the sequence optimizedsequence can be expressed with respect to the theoretical minimumuridine content in the sequence. The term “theoretical minimum uridinecontent” is defined as the uridine content of a nucleic acid sequence asa percentage of the sequence's length after all the codons in thesequence have been replaced with synonymous codon with the lowesturidine content. In some embodiments, the uridine content of thesequence optimized nucleic acid is identical to the theoretical minimumuridine content of the reference sequence (e.g., a wild type sequence).In some aspects, the uridine content of the sequence optimized nucleicacid is about 90%, about 95%, about 100%, about 105%, about 110%, about115%, about 120%, about 125%, about 130%, about 135%, about 140%, about145%, about 150%, about 155%, about 160%, about 165%, about 170%, about175%, about 180%, about 185%, about 190%, about 195% or about 200% ofthe theoretical minimum uridine content of the reference sequence (e.g.,a wild type sequence).

In some embodiments, the uridine content of the sequence optimizednucleic acid is identical to the theoretical minimum uridine content ofthe reference sequence (e.g., a wild type sequence).

The reference nucleic acid sequence (e.g., a wild type sequence) cancomprise uridine clusters which due to their number, size, location,distribution or combinations thereof have negative effects ontranslation. As used herein, the term “uridine cluster” refers to asubsequence in a reference nucleic acid sequence or sequence optimizednucleic sequence with contains a uridine content (usually described as apercentage) which is above a certain threshold. Thus, in certainembodiments, if a subsequence comprises more than about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% uridine content, suchsubsequence would be considered a uridine cluster.

The negative effects of uridine clusters can be, for example, elicitinga TLR7 response. Thus, in some implementations of the nucleic acidsequence optimization methods disclosed herein it is desirable to reducethe number of clusters, size of clusters, location of clusters (e.g.,close to the 5′ and/or 3′ end of a nucleic acid sequence), distancebetween clusters, or distribution of uridine clusters (e.g., a certainpattern of cluster along a nucleic acid sequence, distribution ofclusters with respect to secondary structure elements in the expressedproduct, or distribution of clusters with respect to the secondarystructure of an mRNA).

In some embodiments, the reference nucleic acid sequence comprises atleast one uridine cluster, wherein said uridine cluster is a subsequenceof the reference nucleic acid sequence wherein the percentage of totaluridine nucleobases in said subsequence is above a predeterminedthreshold. In some embodiments, the length of the subsequence is atleast about 10, at least about 15, at least about 20, at least about 25,at least about 30, at least about 35, at least about 40, at least about45, at least about 50, at least about 55, at least about 60, at leastabout 65, at least about 70, at least about 75, at least about 80, atleast about 85, at least about 90, at least about 95, or at least about100 nucleobases. In some embodiments, the subsequence is longer than 100nucleobases. In some embodiments, the threshold is 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24% or 25% uridine content. In some embodiments, thethreshold is above 25%.

For example, an amino acid sequence comprising A, D, G, S and R could beencoded by the nucleic acid sequence GCU, GAU, GGU, AGU, CGU. Althoughsuch sequence does not contain any uridine pairs, triplets, orquadruplets, one third of the nucleobases would be uridines. Such auridine cluster could be removed by using alternative codons, forexample, by using GCC, GAC, GGC, AGC, and CGC, which would contain nouridines.

In other embodiments, the reference nucleic acid sequence comprises atleast one uridine cluster, wherein said uridine cluster is a subsequenceof the reference nucleic acid sequence wherein the percentage of uridinenucleobases of said subsequence as measured using a sliding window thatis above a predetermined threshold. In some embodiments, the length ofthe sliding window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 nucleobases. In some embodiments, the sliding windowis over 40 nucleobases in length. In some embodiments, the threshold is1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% uridine content. In someembodiments, the threshold is above 25%.

In some embodiments, the reference nucleic acid sequence comprises atleast two uridine clusters. In some embodiments, the uridine-modifiedsequence contains fewer uridine-rich clusters than the reference nucleicacid sequence. In some embodiments, the uridine-modified sequencecontains more uridine-rich clusters than the reference nucleic acidsequence. In some embodiments, the uridine-modified sequence containsuridine-rich clusters with are shorter in length than correspondinguridine-rich clusters in the reference nucleic acid sequence. In otherembodiments, the uridine-modified sequence contains uridine-richclusters which are longer in length than the corresponding uridine-richcluster in the reference nucleic acid sequence.

See, Kariko et al. (2005) Immunity 23:165-175; Kormann et al. (2010)Nature Biotechnology 29:154-157; or Sahin et al. (2014) Nature ReviewsDrug Discovery|AOP, published online 19 Sep. 2014m doi:10.1038/nrd4278;all of which are herein incorporated by reference their entireties.

b. Guanine/Cytosine (G/C) Content

A reference nucleic acid sequence can be sequence optimized usingmethods comprising altering the Guanine/Cytosine (G/C) content (absoluteor relative) of the reference nucleic acid sequence. Such optimizationcan comprise altering (e.g., increasing or decreasing) the global G/Ccontent (absolute or relative) of the reference nucleic acid sequence;introducing local changes in G/C content in the reference nucleic acidsequence (e.g., increase or decrease G/C in selected regions orsubsequences in the reference nucleic acid sequence); altering thefrequency, size, and distribution of G/C clusters in the referencenucleic acid sequence, or combinations thereof.

In some embodiments, the sequence optimized nucleic acid encoding Citrincomprises an overall increase in G/C content (absolute or relative)relative to the G/C content (absolute or relative) of the referencenucleic acid sequence. In some embodiments, the overall increase in G/Ccontent (absolute or relative) is at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 100% relative to the G/C content (absolute or relative) of thereference nucleic acid sequence.

In some embodiments, the sequence optimized nucleic acid encoding Citrincomprises an overall decrease in G/C content (absolute or relative)relative to the G/C content of the reference nucleic acid sequence. Insome embodiments, the overall decrease in G/C content (absolute orrelative) is at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 100% relativeto the G/C content (absolute or relative) of the reference nucleic acidsequence.

In some embodiments, the sequence optimized nucleic acid encoding Citrincomprises a local increase in Guanine/Cytosine (G/C) content (absoluteor relative) in a subsequence (i.e., a G/C modified subsequence)relative to the G/C content (absolute or relative) of the correspondingsubsequence in the reference nucleic acid sequence. In some embodiments,the local increase in G/C content (absolute or relative) is by at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 100% relative to the G/C content(absolute or relative) of the corresponding subsequence in the referencenucleic acid sequence.

In some embodiments, the sequence optimized nucleic acid encoding Citrincomprises a local decrease in Guanine/Cytosine (G/C) content (absoluteor relative) in a subsequence (i.e., a G/C modified subsequence)relative to the G/C content (absolute or relative) of the correspondingsubsequence in the reference nucleic acid sequence. In some embodiments,the local decrease in G/C content (absolute or relative) is by at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 100% relative to the G/C content(absolute or relative) of the corresponding subsequence in the referencenucleic acid sequence.

In some embodiments, the G/C content (absolute or relative) is increasedor decreased in a subsequence which is at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100nucleobases in length.

In some embodiments, the G/C content (absolute or relative) is increasedor decreased in a subsequence which is at least about 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, or 1000 nucleobases in length.

In some embodiments, the G/C content (absolute or relative) is increasedor decreased in a subsequence which is at least about 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700,3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900,5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100,6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300,7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500,8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700,9800, 9900, or 10000 nucleobases in length. The increases or decreasesin G and C content (absolute or relative) described herein can beconducted by replacing synonymous codons with low G/C content withsynonymous codons having higher G/C content, or vice versa. For example,L has 6 synonymous codons: two of them have 2 G/C (CUC, CUG), 3 have asingle G/C (UUG, CUU, CUA), and one has no G/C (UUA). So if thereference nucleic acid had a CUC codon in a certain position, G/Ccontent at that position could be reduced by replacing CUC with any ofthe codons having a single G/C or the codon with no G/C.

See, U.S. Publ. Nos. US20140228558, US20050032730 A1; Gustafsson et al.(2012) Protein Expression and Purification 83: 37-46; all of which areincorporated herein by reference in their entireties.

c. Codon Frequency—Codon Usage Bias

Numerous codon optimization methods known in the art are based on thesubstitution of codons in a reference nucleic acid sequence with codonshaving higher frequencies. Thus, in some embodiments, a nucleic acidsequence encoding Citrin disclosed herein can be sequence optimizedusing methods comprising the use of modifications in the frequency ofuse of one or more codons relative to other synonymous codons in thesequence optimized nucleic acid with respect to the frequency of use inthe non-codon optimized sequence.

As used herein, the term “codon frequency” refers to codon usage bias,i.e., the differences in the frequency of occurrence of synonymouscodons in coding DNA/RNA. It is generally acknowledged that codonpreferences reflect a balance between mutational biases and naturalselection for translational optimization. Optimal codons help to achievefaster translation rates and high accuracy. As a result of thesefactors, translational selection is expected to be stronger in highlyexpressed genes. In the field of bioinformatics and computationalbiology, many statistical methods have been proposed and used to analyzecodon usage bias. See, e.g., Comeron & Aguade (1998) J. Mol. Evol. 47:268-74. Methods such as the ‘frequency of optimal codons’ (Fop) (Ikemura(1981) J. Mol. Biol. 151 (3): 389-409), the Relative Codon Adaptation(RCA) (Fox & Eril (2010) DNA Res. 17 (3): 185-96) or the ‘CodonAdaptation Index’ (CAI) (Sharp & Li (1987) Nucleic Acids Res. 15 (3):1281-95) are used to predict gene expression levels, while methods suchas the ‘effective number of codons’ (Nc) and Shannon entropy frominformation theory are used to measure codon usage evenness.Multivariate statistical methods, such as correspondence analysis andprincipal component analysis, are widely used to analyze variations incodon usage among genes (Suzuki et al. (2008) DNA Res. 15 (6): 357-65;Sandhu et al., In Silico Biol. 2008; 8(2):187-92).

The nucleic acid sequence encoding a Citrin polypeptide disclosed herein(e.g., a wild type nucleic acid sequence, a mutant nucleic acidsequence, a chimeric nucleic sequence, etc. which can be, for example,an mRNA), can be codon optimized using methods comprising substitutingat least one codon in the reference nucleic acid sequence with analternative codon having a higher or lower codon frequency in thesynonymous codon set; wherein the resulting sequence optimized nucleicacid has at least one optimized property with respect to the referencenucleic acid sequence.

In some embodiments, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 99%,or 100% of the codons in the reference nucleic acid sequence encodingCitrin are substituted with alternative codons, each alternative codonhaving a codon frequency higher than the codon frequency of thesubstituted codon in the synonymous codon set.

In some embodiments, at least one codon in the reference nucleic acidsequence encoding Citrin is substituted with an alternative codon havinga codon frequency higher than the codon frequency of the substitutedcodon in the synonymous codon set, and at least one codon in thereference nucleic acid sequence is substituted with an alternative codonhaving a codon frequency lower than the codon frequency of thesubstituted codon in the synonymous codon set.

In some embodiments, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, or at least about 75% of the codons in the referencenucleic acid sequence encoding Citrin are substituted with alternativecodons, each alternative codon having a codon frequency higher than thecodon frequency of the substituted codon in the synonymous codon set.

In some embodiments, at least one alternative codon having a highercodon frequency has the highest codon frequency in the synonymous codonset. In other embodiments, all alternative codons having a higher codonfrequency have the highest codon frequency in the synonymous codon set.

In some embodiments, at least one alternative codon having a lower codonfrequency has the lowest codon frequency in the synonymous codon set. Insome embodiments, all alternative codons having a higher codon frequencyhave the highest codon frequency in the synonymous codon set.

In some specific embodiments, at least one alternative codon has thesecond highest, the third highest, the fourth highest, the fifth highestor the sixth highest frequency in the synonymous codon set. In somespecific embodiments, at least one alternative codon has the secondlowest, the third lowest, the fourth lowest, the fifth lowest, or thesixth lowest frequency in the synonymous codon set.

Optimization based on codon frequency can be applied globally, asdescribed above, or locally to the reference nucleic acid sequenceencoding a Citrin polypeptide. In some embodiments, when appliedlocally, regions of the reference nucleic acid sequence can modifiedbased on codon frequency, substituting all or a certain percentage ofcodons in a certain subsequence with codons that have higher or lowerfrequencies in their respective synonymous codon sets. Thus, in someembodiments, at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 99%, or 100% ofthe codons in a subsequence of the reference nucleic acid sequence aresubstituted with alternative codons, each alternative codon having acodon frequency higher than the codon frequency of the substituted codonin the synonymous codon set.

In some embodiments, at least one codon in a subsequence of thereference nucleic acid sequence encoding a Citrin polypeptide issubstituted with an alternative codon having a codon frequency higherthan the codon frequency of the substituted codon in the synonymouscodon set, and at least one codon in a subsequence of the referencenucleic acid sequence is substituted with an alternative codon having acodon frequency lower than the codon frequency of the substituted codonin the synonymous codon set.

In some embodiments, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, or at least about 75% of the codons in a subsequenceof the reference nucleic acid sequence encoding a Citrin polypeptide aresubstituted with alternative codons, each alternative codon having acodon frequency higher than the codon frequency of the substituted codonin the synonymous codon set.

In some embodiments, at least one alternative codon substituted in asubsequence of the reference nucleic acid sequence encoding a Citrinpolypeptide and having a higher codon frequency has the highest codonfrequency in the synonymous codon set. In other embodiments, allalternative codons substituted in a subsequence of the reference nucleicacid sequence and having a lower codon frequency have the lowest codonfrequency in the synonymous codon set.

In some embodiments, at least one alternative codon substituted in asubsequence of the reference nucleic acid sequence encoding a Citrinpolypeptide and having a lower codon frequency has the lowest codonfrequency in the synonymous codon set. In some embodiments, allalternative codons substituted in a subsequence of the reference nucleicacid sequence and having a higher codon frequency have the highest codonfrequency in the synonymous codon set.

In specific embodiments, a sequence optimized nucleic acid encoding aCitrin polypeptide can comprise a subsequence having an overall codonfrequency higher or lower than the overall codon frequency in thecorresponding subsequence of the reference nucleic acid sequence at aspecific location, for example, at the 5′ end or 3′ end of the sequenceoptimized nucleic acid, or within a predetermined distance from thoseregion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 codons fromthe 5′ end or 3′ end of the sequence optimized nucleic acid). In someembodiments, an sequence optimized nucleic acid encoding a Citrinpolypeptide can comprise more than one subsequence having an overallcodon frequency higher or lower than the overall codon frequency in thecorresponding subsequence of the reference nucleic acid sequence. Askilled artisan would understand that subsequences with overall higheror lower overall codon frequencies can be organized in innumerablepatterns, depending on whether the overall codon frequency is higher orlower, the length of the subsequence, the distance between subsequences,the location of the subsequences, etc.

See, U.S. Pat. Nos. U.S. Pat. Nos. 5,082,767, 8,126,653, 7,561,973,8,401,798; U.S. Publ. No. US 20080046192, US 20080076161; Int'l. Publ.No. WO2000018778; Welch et al. (2009) PLoS ONE 4(9): e7002; Gustafssonet al. (2012) Protein Expression and Purification 83: 37-46; Chung etal. (2012) BMC Systems Biology 6:134; all of which are incorporatedherein by reference in their entireties.

d. Destabilizing Motif Substitution

There is a variety of motifs that can affect sequence optimization,which fall into various non-exclusive categories, for example:

(i) Primary sequence based motifs: Motifs defined by a simplearrangement of nucleotides.

(ii) Structural motifs: Motifs encoded by an arrangement of nucleotidesthat tends to form a certain secondary structure.

(iii) Local motifs: Motifs encoded in one contiguous subsequence.

(iv) Distributed motifs: Motifs encoded in two or more disjointsubsequences.

(v) Advantageous motifs: Motifs which improve nucleotide structure orfunction.

(vi) Disadvantageous motifs: Motifs with detrimental effects onnucleotide structure or function.

There are many motifs that fit into the category of disadvantageousmotifs. Some examples include, for example, restriction enzyme motifs,which tend to be relatively short, exact sequences such as therestriction site motifs for Xba1 (TCTAGA), EcoRI (GAATTC), EcoRII(CCWGG, wherein W means A or T, per the IUPAC ambiguity codes), orHindIII (AAGCTT); enzyme sites, which tend to be longer and based onconsensus not exact sequence, such in the T7 RNA polymerase(GnnnnWnCRnCTCnCnnWnD, wherein n means any nucleotide, R means A or G, Wmeans A or T, D means A or G or T but not C); structural motifs, such asGGGG repeats (Kim et al. (1991) Nature 351(6324):331-2); or other motifssuch as CUG-triplet repeats (Querido et al. (2014) J. Cell Sci.124:1703-1714).

Accordingly, the nucleic acid sequence encoding a Citrin polypeptidedisclosed herein can be sequence optimized using methods comprisingsubstituting at least one destabilizing motif in a reference nucleicacid sequence, and removing such disadvantageous motif or replacing itwith an advantageous motif

In some embodiments, the optimization process comprises identifyingadvantageous and/or disadvantageous motifs in the reference nucleicsequence, wherein such motifs are, e.g., specific subsequences that cancause a loss of stability in the reference nucleic acid sequence prioror during the optimization process. For example, substitution ofspecific bases during optimization can generate a subsequence (motif)recognized by a restriction enzyme. Accordingly, during the optimizationprocess the appearance of disadvantageous motifs can be monitored bycomparing the sequence optimized sequence with a library of motifs knownto be disadvantageous. Then, the identification of disadvantageousmotifs could be used as a post-hoc filter, i.e., to determine whether acertain modification which potentially could be introduced in thereference nucleic acid sequence should be actually implemented or not.

In some embodiments, the identification of disadvantageous motifs can beused prior to the application of the sequence optimization methodsdisclosed herein, i.e., the identification of motifs in the referencenucleic acid sequence encoding a Citrin polypeptide and theirreplacement with alternative nucleic acid sequences can be used as apreprocessing step, for example, before uridine reduction. In otherembodiments, the identification of disadvantageous motifs and theirremoval is used as an additional sequence optimization techniqueintegrated in a multiparametric nucleic acid optimization methodcomprising two or more of the sequence optimization methods disclosedherein. When used in this fashion, a disadvantageous motif identifiedduring the optimization process would be removed, for example, bysubstituting the lowest possible number of nucleobases in order topreserve as closely as possible the original design principle(s) (e.g.,low U, high frequency, etc.).

See, e.g., U.S. Publ. Nos. US20140228558, US20050032730, orUS20140228558, which are herein incorporated by reference in theirentireties.

e. Limited Codon Set Optimization

In some particular embodiments, sequence optimization of a referencenucleic acid sequence encoding a Citrin polypeptide can be conductedusing a limited codon set, e.g., a codon set wherein less than thenative number of codons is used to encode the 20 natural amino acids, asubset of the 20 natural amino acids, or an expanded set of amino acidsincluding, for example, non-natural amino acids.

The genetic code is highly similar among all organisms and can beexpressed in a simple table with 64 entries which would encode the 20standard amino acids involved in protein translation plus start and stopcodons. The genetic code is degenerate, i.e., in general, more than onecodon specifies each amino acid. For example, the amino acid leucine isspecified by the UUA, UUG, CUU, CUC, CUA, or CUG codons, while the aminoacid serine is specified by UCA, UCG, UCC, UCU, AGU, or AGC codons(difference in the first, second, or third position). Native geneticcodes comprise 62 codons encoding naturally occurring amino acids. Thus,in some embodiments of the methods disclosed herein optimized codon sets(genetic codes) comprising less than 62 codons to encode 20 amino acidscan comprise 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47,46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 codons.

In some embodiments, the limited codon set comprises less than 20codons. For example, if a protein contains less than 20 types of aminoacids, such protein could be encoded by a codon set with less than 20codons. Accordingly, in some embodiments, an optimized codon setcomprises as many codons as different types of amino acids are presentin the protein encoded by the reference nucleic acid sequence. In someembodiments, the optimized codon set comprises 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or even 1 codon.

In some embodiments, at least one amino acid selected from the groupconsisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Phe, Pro, Ser, Thr, Tyr, and Val, i.e., amino acids which arenaturally encoded by more than one codon, is encoded with less codonsthan the naturally occurring number of synonymous codons. For example,in some embodiments, Ala can be encoded in the sequence optimizednucleic acid by 3, 2 or 1 codons; Cys can be encoded in the sequenceoptimized nucleic acid by 1 codon; Asp can be encoded in the sequenceoptimized nucleic acid by 1 codon; Glu can be encoded in the sequenceoptimized nucleic acid by 1 codon; Phe can be encoded in the sequenceoptimized nucleic acid by 1 codon; Gly can be encoded in the sequenceoptimized nucleic acid by 3 codons, 2 codons or 1 codon; His can beencoded in the sequence optimized nucleic acid by 1 codon; Ile can beencoded in the sequence optimized nucleic acid by 2 codons or 1 codon;Lys can be encoded in the sequence optimized nucleic acid by 1 codon;Leu can be encoded in the sequence optimized nucleic acid by 5 codons, 4codons, 3 codons, 2 codons or 1 codon; Asn can be encoded in thesequence optimized nucleic acid by 1 codon; Pro can be encoded in thesequence optimized nucleic acid by 3 codons, 2 codons, or 1 codon; Glncan be encoded in the sequence optimized nucleic acid by 1 codon; Argcan be encoded in the sequence optimized nucleic acid by 5 codons, 4codons, 3 codons, 2 codons, or 1 codon; Ser can be encoded in thesequence optimized nucleic acid by 5 codons, 4 codons, 3 codons, 2codons, or 1 codon; Thr can be encoded in the sequence optimized nucleicacid by 3 codons, 2 codons, or 1 codon; Val can be encoded in thesequence optimized nucleic acid by 3 codons, 2 codons, or 1 codon; and,Tyr can be encoded in the sequence optimized nucleic acid by 1 codon.

In some embodiments, at least one amino acid selected from the groupconsisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Phe, Pro, Ser, Thr, Tyr, and Val, i.e., amino acids which arenaturally encoded by more than one codon, is encoded by a single codonin the limited codon set.

In some specific embodiments, the sequence optimized nucleic acid is aDNA and the limited codon set consists of 20 codons, wherein each codonencodes one of 20 amino acids. In some embodiments, the sequenceoptimized nucleic acid is a DNA and the limited codon set comprises atleast one codon selected from the group consisting of GCT, GCC, GCA, andGCG; at least a codon selected from the group consisting of CGT, CGC,CGA, CGG, AGA, and AGG; at least a codon selected from AAT or ACC; atleast a codon selected from GAT or GAC; at least a codon selected fromTGT or TGC; at least a codon selected from CAA or CAG; at least a codonselected from GAA or GAG; at least a codon selected from the groupconsisting of GGT, GGC, GGA, and GGG; at least a codon selected from CATor CAC; at least a codon selected from the group consisting of ATT, ATC,and ATA; at least a codon selected from the group consisting of TTA,TTG, CTT, CTC, CTA, and CTG; at least a codon selected from AAA or AAG;an ATG codon; at least a codon selected from TTT or TTC; at least acodon selected from the group consisting of CCT, CCC, CCA, and CCG; atleast a codon selected from the group consisting of TCT, TCC, TCA, TCG,AGT, and AGC; at least a codon selected from the group consisting ofACT, ACC, ACA, and ACG; a TGG codon; at least a codon selected from TATor TAC; and, at least a codon selected from the group consisting of GTT,GTC, GTA, and GTG.

In other embodiments, the sequence optimized nucleic acid is an RNA(e.g., an mRNA) and the limited codon set consists of 20 codons, whereineach codon encodes one of 20 amino acids. In some embodiments, thesequence optimized nucleic acid is an RNA and the limited codon setcomprises at least one codon selected from the group consisting of GCU,GCC, GCA, and GCG; at least a codon selected from the group consistingof CGU, CGC, CGA, CGG, AGA, and AGG; at least a codon selected from AAUor ACC; at least a codon selected from GAU or GAC; at least a codonselected from UGU or UGC; at least a codon selected from CAA or CAG; atleast a codon selected from GAA or GAG; at least a codon selected fromthe group consisting of GGU, GGC, GGA, and GGG; at least a codonselected from CAU or CAC; at least a codon selected from the groupconsisting of AUU, AUC, and AUA; at least a codon selected from thegroup consisting of UUA, UUG, CUU, CUC, CUA, and CUG; at least a codonselected from AAA or AAG; an AUG codon; at least a codon selected fromUUU or UUC; at least a codon selected from the group consisting of CCU,CCC, CCA, and CCG; at least a codon selected from the group consistingof UCU, UCC, UCA, UCG, AGU, and AGC; at least a codon selected from thegroup consisting of ACU, ACC, ACA, and ACG; a UGG codon; at least acodon selected from UAU or UAC; and, at least a codon selected from thegroup consisting of GUU, GUC, GUA, and GUG.

In some specific embodiments, the limited codon set has been optimizedfor in vivo expression of a sequence optimized nucleic acid (e.g., asynthetic mRNA) following administration to a certain tissue or cell.

In some embodiments, the optimized codon set (e.g., a 20 codon setencoding 20 amino acids) complies at least with one of the followingproperties:

-   -   (i) the optimized codon set has a higher average G/C content        than the original or native codon set; or,    -   (ii) the optimized codon set has a lower average U content than        the original or native codon set; or,    -   (iii) the optimized codon set is composed of codons with the        highest frequency; or,    -   (iv) the optimized codon set is composed of codons with the        lowest frequency; or,    -   (v) a combination thereof.

In some specific embodiments, at least one codon in the optimized codonset has the second highest, the third highest, the fourth highest, thefifth highest or the sixth highest frequency in the synonymous codonset. In some specific embodiments, at least one codon in the optimizedcodon has the second lowest, the third lowest, the fourth lowest, thefifth lowest, or the sixth lowest frequency in the synonymous codon set.

As used herein, the term “native codon set” refers to the codon set usednatively by the source organism to encode the reference nucleic acidsequence. As used herein, the term “original codon set” refers to thecodon set used to encode the reference nucleic acid sequence before thebeginning of sequence optimization, or to a codon set used to encode anoptimized variant of the reference nucleic acid sequence at thebeginning of a new optimization iteration when sequence optimization isapplied iteratively or recursively.

In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in thecodon set are those with the highest frequency. In other embodiments,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are thosewith the lowest frequency.

In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in thecodon set are those with the highest uridine content. In someembodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set arethose with the lowest uridine content.

In some embodiments, the average G/C content (absolute or relative) ofthe codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the averageG/C content (absolute or relative) of the original codon set. In someembodiments, the average G/C content (absolute or relative) of the codonset is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100% lower than the average G/C content(absolute or relative) of the original codon set.

In some embodiments, the uracil content (absolute or relative) of thecodon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the average uracilcontent (absolute or relative) of the original codon set. In someembodiments, the uracil content (absolute or relative) of the codon setis 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 100% lower than the average uracil content(absolute or relative) of the original codon set.

See also U.S. Appl. Publ. No. 2011/0082055, and Int'l. Publ. No.WO2000018778, both of which are incorporated herein by reference intheir entireties.

8. Characterization of Sequence Optimized Nucleic Acids

In some embodiments of the invention, the polynucleotide (e.g., a RNA,e.g., an mRNA) comprising a sequence optimized nucleic acid disclosedherein encoding a Citrin polypeptide can be tested to determine whetherat least one nucleic acid sequence property (e.g., stability whenexposed to nucleases) or expression property has been improved withrespect to the non-sequence optimized nucleic acid.

As used herein, “expression property” refers to a property of a nucleicacid sequence either in vivo (e.g., translation efficacy of a syntheticmRNA after administration to a subject in need thereof) or in vitro(e.g., translation efficacy of a synthetic mRNA tested in an in vitromodel system). Expression properties include but are not limited to theamount of protein produced by an mRNA encoding a Citrin polypeptideafter administration, and the amount of soluble or otherwise functionalprotein produced. In some embodiments, sequence optimized nucleic acidsdisclosed herein can be evaluated according to the viability of thecells expressing a protein encoded by a sequence optimized nucleic acidsequence (e.g., a RNA, e.g., an mRNA) encoding a Citrin polypeptidedisclosed herein.

In a particular embodiment, a plurality of sequence optimized nucleicacids disclosed herein (e.g., a RNA, e.g., an mRNA) containing codonsubstitutions with respect to the non-optimized reference nucleic acidsequence can be characterized functionally to measure a property ofinterest, for example an expression property in an in vitro modelsystem, or in vivo in a target tissue or cell.

a. Optimization of Nucleic Acid Sequence Intrinsic Properties

In some embodiments of the invention, the desired property of thepolynucleotide is an intrinsic property of the nucleic acid sequence.For example, the nucleotide sequence (e.g., a RNA, e.g., an mRNA) can besequence optimized for in vivo or in vitro stability. In someembodiments, the nucleotide sequence can be sequence optimized forexpression in a particular target tissue or cell. In some embodiments,the nucleic acid sequence is sequence optimized to increase its plasmahalf life by preventing its degradation by endo and exonucleases.

In other embodiments, the nucleic acid sequence is sequence optimized toincrease its resistance to hydrolysis in solution, for example, tolengthen the time that the sequence optimized nucleic acid or apharmaceutical composition comprising the sequence optimized nucleicacid can be stored under aqueous conditions with minimal degradation.

In other embodiments, the sequence optimized nucleic acid can beoptimized to increase its resistance to hydrolysis in dry storageconditions, for example, to lengthen the time that the sequenceoptimized nucleic acid can be stored after lyophilization with minimaldegradation.

b. Nucleic Acids Sequence Optimized for Protein Expression

In some embodiments of the invention, the desired property of thepolynucleotide is the level of expression of a Citrin polypeptideencoded by a sequence optimized sequence disclosed herein. Proteinexpression levels can be measured using one or more expression systems.In some embodiments, expression can be measured in cell culture systems,e.g., CHO cells or HEK293 cells. In some embodiments, expression can bemeasured using in vitro expression systems prepared from extracts ofliving cells, e.g., rabbit reticulocyte lysates, or in vitro expressionsystems prepared by assembly of purified individual components. In otherembodiments, the protein expression is measured in an in vivo system,e.g., mouse, rabbit, monkey, etc.

In some embodiments, protein expression in solution form can bedesirable. Accordingly, in some embodiments, a reference sequence can besequence optimized to yield a sequence optimized nucleic acid sequencehaving optimized levels of expressed proteins in soluble form. Levels ofprotein expression and other properties such as solubility, levels ofaggregation, and the presence of truncation products (i.e., fragmentsdue to proteolysis, hydrolysis, or defective translation) can bemeasured according to methods known in the art, for example, usingelectrophoresis (e.g., native or SDS-PAGE) or chromatographic methods(e.g., HPLC, size exclusion chromatography, etc.).

c. Optimization of Target Tissue or Target Cell Viability

In some embodiments, the expression of heterologous therapeutic proteinsencoded by a nucleic acid sequence can have deleterious effects in thetarget tissue or cell, reducing protein yield, or reducing the qualityof the expressed product (e.g., due to the presence of protein fragmentsor precipitation of the expressed protein in inclusion bodies), orcausing toxicity.

Accordingly, in some embodiments of the invention, the sequenceoptimization of a nucleic acid sequence disclosed herein, e.g., anucleic acid sequence encoding a Citrin polypeptide, can be used toincrease the viability of target cells expressing the protein encoded bythe sequence optimized nucleic acid.

Heterologous protein expression can also be deleterious to cellstransfected with a nucleic acid sequence for autologous or heterologoustransplantation. Accordingly, in some embodiments of the presentdisclosure the sequence optimization of a nucleic acid sequencedisclosed herein can be used to increase the viability of target cellsexpressing the protein encoded by the sequence optimized nucleic acidsequence. Changes in cell or tissue viability, toxicity, and otherphysiological reaction can be measured according to methods known in theart.

d. Reduction of Immune and/or Inflammatory Response

In some cases, the administration of a sequence optimized nucleic acidencoding Citrin polypeptide or a functional fragment thereof can triggeran immune response, which could be caused by (i) the therapeutic agent(e.g., an mRNA encoding a Citrin polypeptide), or (ii) the expressionproduct of such therapeutic agent (e.g., the Citrin polypeptide encodedby the mRNA), or (iv) a combination thereof. Accordingly, in someembodiments of the present disclosure the sequence optimization ofnucleic acid sequence (e.g., an mRNA) disclosed herein can be used todecrease an immune or inflammatory response triggered by theadministration of a nucleic acid encoding a Citrin polypeptide or by theexpression product of Citrin encoded by such nucleic acid.

In some aspects, an inflammatory response can be measured by detectingincreased levels of one or more inflammatory cytokines using methodsknown in the art, e.g., ELISA. The term “inflammatory cytokine” refersto cytokines that are elevated in an inflammatory response. Examples ofinflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine(C-X-C motif) ligand 1; also known as GROα, interferon-γ (IFNγ), tumornecrosis factor α (TNFα), interferon γ-induced protein 10 (IP-10), orgranulocyte-colony stimulating factor (G-CSF). The term inflammatorycytokines includes also other cytokines associated with inflammatoryresponses known in the art, e.g., interleukin-1 (IL-1), interleukin-8(IL-8), interleukin-12 (IL-12), interleukin-13 (Il-13), interferon α(IFN-α), etc.

9. Modified Nucleotide Sequences Encoding Citrin Polypeptides

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a chemically modified nucleobase, e.g.,5-methoxyuracil. In some embodiments, the mRNA is a uracil-modifiedsequence comprising an ORF encoding a Citrin polypeptide, wherein themRNA comprises a chemically modified nucleobase, e.g., 5-methoxyuracil.

In certain aspects of the invention, when the 5-methoxyuracil base isconnected to a ribose sugar, as it is in polynucleotides, the resultingmodified nucleoside or nucleotide is referred to as 5-methoxyuridine. Insome embodiments, uracil in the polynucleotide is at least about 25%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least 90%, at least 95%,at least 99%, or about 100% 5-methoxyuracil. In one embodiment, uracilin the polynucleotide is at least 95% 5-methoxyuracil. In anotherembodiment, uracil in the polynucleotide is 100% 5-methoxyuracil.

In embodiments where uracil in the polynucleotide is at least 95%5-methoxyuracil, overall uracil content can be adjusted such that anmRNA provides suitable protein expression levels while inducing littleto no immune response. In some embodiments, the uracil content of theORF is between about 105% and about 145%, about 105% and about 140%,about 110% and about 140%, about 110% and about 145%, about 115% andabout 135%, about 105% and about 135%, about 110% and about 135%, about115% and about 145%, or about 115% and about 140% of the theoreticalminimum uracil content in the corresponding wild-type ORF (% Utm). Inother embodiments, the uracil content of the ORF is between about 117%and about 134% or between 118% and 132% of the % U_(TM). In someembodiments, the uracil content of the ORF encoding a Citrin polypeptideis about 115%, about 120%, about 125%, about 130%, about 135%, about140%, about 145%, or about 150% of the % Utm. In this context, the term“uracil” can refer to 5-methoxyuracil and/or naturally occurring uracil.

In some embodiments, the uracil content in the ORF of the mRNA encodinga Citrin polypeptide of the invention is less than about 50%, about 40%,about 30%, about 20% or about 15% of the total nucleobase content in theORF. In some embodiments, the uracil content in the ORF is between about15% and about 25% of the total nucleobase content in the ORF. In otherembodiments, the uracil content in the ORF is between about 20% andabout 30% of the total nucleobase content in the ORF. In one embodiment,the uracil content in the ORF of the mRNA encoding a Citrin polypeptideis less than about 30% of the total nucleobase content in the openreading frame. In this context, the term “uracil” can refer to5-methoxyuracil and/or naturally occurring uracil.

In further embodiments, the ORF of the mRNA encoding a Citrinpolypeptide having 5-methoxyuracil and adjusted uracil content hasincreased Cytosine (C), Guanine (G), or Guanine/Cytosine (G/C) content(absolute or relative). In some embodiments, the overall increase in C,G, or G/C content (absolute or relative) of the ORF is at least about2%, at least about 3%, at least about 4%, at least about 5%, at leastabout 6%, at least about 7%, at least about 10%, at least about 15%, atleast about 20%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, or at least about 100% relative to the G/C content(absolute or relative) of the wild-type ORF. In some embodiments, the G,the C, or the G/C content in the ORF is less than about 100%, less thanabout 90%, less than about 85%, or less than about 80% of thetheoretical maximum G, C, or G/C content of the corresponding wild typenucleotide sequence encoding the Citrin polypeptide (% G_(TMX); %C_(TMX), or % G_(TMX)). In other embodiments, the G, the C, or the G/Ccontent in the ORF is between about 70% and about 80%, between about 71%and about 79%, between about 71% and about 78%, or between about 71% andabout 77% of the % G_(TMX), % G_(TMX), or % G/C_(TMX). In someembodiments, the increases in G and/or C content (absolute or relative)described herein can be conducted by replacing synonymous codons withlow G, C, or G/C content with synonymous codons having higher G, C, orG/C content. In other embodiments, the increase in G and/or C content(absolute or relative) is conducted by replacing a codon ending with Uwith a synonymous codon ending with G or C.

In further embodiments, the ORF of the mRNA encoding a Citrinpolypeptide of the invention comprises 5-methoxyuracil and has anadjusted uracil content containing less uracil pairs (UU) and/or uraciltriplets (UUU) and/or uracil quadruplets (UUUU) than the correspondingwild-type nucleotide sequence encoding the Citrin polypeptide. In someembodiments, the ORF of the mRNA encoding a Citrin polypeptide of theinvention contains no uracil pairs and/or uracil triplets and/or uracilquadruplets. In some embodiments, uracil pairs and/or uracil tripletsand/or uracil quadruplets are reduced below a certain threshold, e.g.,no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 occurrences in the ORF of the mRNA encoding the Citrinpolypeptide. In a particular embodiment, the ORF of the mRNA encodingthe Citrin polypeptide of the invention contains less than 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1non-phenylalanine uracil pairs and/or triplets. In another embodiment,the ORF of the mRNA encoding the Citrin polypeptide contains nonon-phenylalanine uracil pairs and/or triplets.

In further embodiments, the ORF of the mRNA encoding a Citrinpolypeptide of the invention comprises 5-methoxyuracil and has anadjusted uracil content containing less uracil-rich clusters than thecorresponding wild-type nucleotide sequence encoding the Citrinpolypeptide. In some embodiments, the ORF of the mRNA encoding theCitrin polypeptide of the invention contains uracil-rich clusters thatare shorter in length than corresponding uracil-rich clusters in thecorresponding wild-type nucleotide sequence encoding the Citrinpolypeptide.

In further embodiments, alternative lower frequency codons are employed.At least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 99%, or 100% of the codonsin the Citrin polypeptide-encoding ORF of the 5-methoxyuracil-comprisingmRNA are substituted with alternative codons, each alternative codonhaving a codon frequency lower than the codon frequency of thesubstituted codon in the synonymous codon set. The ORF also has adjusteduracil content, as described above. In some embodiments, at least onecodon in the ORF of the mRNA encoding the Citrin polypeptide issubstituted with an alternative codon having a codon frequency lowerthan the codon frequency of the substituted codon in the synonymouscodon set.

In some embodiments, the adjusted uracil content, Citrinpolypeptide-encoding ORF of the 5-methoxyuracil-comprising mRNA exhibitsexpression levels of Citrin when administered to a mammalian cell thatare higher than expression levels of Citrin from the correspondingwild-type mRNA. In other embodiments, the expression levels of Citrinwhen administered to a mammalian cell are increased relative to acorresponding mRNA containing at least 95% 5-methoxyuracil and having auracil content of about 160%, about 170%, about 180%, about 190%, orabout 200% of the theoretical minimum. In yet other embodiments, theexpression levels of Citrin when administered to a mammalian cell areincreased relative to a corresponding mRNA, wherein at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, or about 100% of uracils are 1-methylpseudouracil orpseudouracils. In some embodiments, the mammalian cell is a mouse cell,a rat cell, or a rabbit cell. In other embodiments, the mammalian cellis a monkey cell or a human cell. In some embodiments, the human cell isa HeLa cell, a BJ fibroblast cell, or a peripheral blood mononuclearcell (PBMC). In some embodiments, Citrin is expressed when the mRNA isadministered to a mammalian cell in vivo. In some embodiments, the mRNAis administered to mice, rabbits, rats, monkeys, or humans. In oneembodiment, mice are null mice. In some embodiments, the mRNA isadministered to mice in an amount of about 0.01 mg/kg, about 0.05 mg/kg,about 0.1 mg/kg, or about 0.15 mg/kg. In some embodiments, the mRNA isadministered intravenously or intramuscularly. In other embodiments, theCitrin polypeptide is expressed when the mRNA is administered to amammalian cell in vitro. In some embodiments, the expression isincreased by at least about 2-fold, at least about 5-fold, at leastabout 10-fold, at least about 50-fold, at least about 500-fold, at leastabout 1500-fold, or at least about 3000-fold. In other embodiments, theexpression is increased by at least about 10%, about 20%, about 30%,about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about100%.

In some embodiments, adjusted uracil content, Citrinpolypeptide-encoding ORF of the 5-methoxyuracil-comprising mRNA exhibitsincreased stability. In some embodiments, the mRNA exhibits increasedstability in a cell relative to the stability of a correspondingwild-type mRNA under the same conditions. In some embodiments, the mRNAexhibits increased stability including resistance to nucleases, thermalstability, and/or increased stabilization of secondary structure. Insome embodiments, increased stability exhibited by the mRNA is measuredby determining the half-life of the mRNA (e.g., in a plasma, cell, ortissue sample) and/or determining the area under the curve (AUC) of theprotein expression by the mRNA over time (e.g., in vitro or in vivo). AnmRNA is identified as having increased stability if the half-life and/orthe AUC is greater than the half-life and/or the AUC of a correspondingwild-type mRNA under the same conditions.

In some embodiments, the mRNA of the present invention induces adetectably lower immune response (e.g., innate or acquired) relative tothe immune response induced by a corresponding wild-type mRNA under thesame conditions. In other embodiments, the mRNA of the presentdisclosure induces a detectably lower immune response (e.g., innate oracquired) relative to the immune response induced by an mRNA thatencodes for a Citrin polypeptide but does not comprise 5-methoxyuracilunder the same conditions, or relative to the immune response induced byan mRNA that encodes for a Citrin polypeptide and that comprises5-methoxyuracil but that does not have adjusted uracil content under thesame conditions. The innate immune response can be manifested byincreased expression of pro-inflammatory cytokines, activation ofintracellular PRRs (RIG-I, MDAS, etc.), cell death, and/or terminationor reduction in protein translation. In some embodiments, a reduction inthe innate immune response can be measured by expression or activitylevel of Type 1 interferons (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε,IFN-τ, IFN-ω, and IFN-ζ) or the expression of interferon-regulated genessuch as the toll-like receptors (e.g., TLR7 and TLR8), and/or bydecreased cell death following one or more administrations of the mRNAof the invention into a cell.

In some embodiments, the expression of Type-1 interferons by a mammaliancell in response to the mRNA of the present disclosure is reduced by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greaterthan 99% relative to a corresponding wild-type mRNA, to an mRNA thatencodes a Citrin polypeptide but does not comprise 5-methoxyuracil, orto an mRNA that encodes a Citrin polypeptide and that comprises5-methoxyuracil but that does not have adjusted uracil content. In someembodiments, the interferon is IFN-β. In some embodiments, cell deathfrequency cased by administration of mRNA of the present disclosure to amammalian cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% lessthan the cell death frequency observed with a corresponding wild-typemRNA, an mRNA that encodes for a Citrin polypeptide but does notcomprise 5-methoxyuracil, or an mRNA that encodes for a Citrinpolypeptide and that comprises 5-methoxyuracil but that does not haveadjusted uracil content. In some embodiments, the mammalian cell is a BJfibroblast cell. In other embodiments, the mammalian cell is asplenocyte. In some embodiments, the mammalian cell is that of a mouseor a rat. In other embodiments, the mammalian cell is that of a human.In one embodiment, the mRNA of the present disclosure does notsubstantially induce an innate immune response of a mammalian cell intowhich the mRNA is introduced.

In some embodiments, the polynucleotide is an mRNA that comprises an ORFthat encodes a Citrin polypeptide, wherein uracil in the mRNA is atleast about 95% 5-methoxyuracil, wherein the uracil content of the ORFis between about 115% and about 135% of the theoretical minimum uracilcontent in the corresponding wild-type ORF, and wherein the uracilcontent in the ORF encoding the Citrin polypeptide is less than about30% of the total nucleobase content in the ORF. In some embodiments, theORF that encodes the Citrin polypeptide is further modified to increaseG/C content of the ORF (absolute or relative) by at least about 40%, ascompared to the corresponding wild-type ORF. In yet other embodiments,the ORF encoding the Citrin polypeptide contains less than 20non-phenylalanine uracil pairs and/or triplets. In some embodiments, atleast one codon in the ORF of the mRNA encoding the Citrin polypeptideis further substituted with an alternative codon having a codonfrequency lower than the codon frequency of the substituted codon in thesynonymous codon set. In some embodiments, the expression of the Citrinpolypeptide encoded by an mRNA comprising an ORF wherein uracil in themRNA is at least about 95% 5-methoxyuracil, and wherein the uracilcontent of the ORF is between about 115% and about 135% of thetheoretical minimum uracil content in the corresponding wild-type ORF,is increased by at least about 10-fold when compared to expression ofthe Citrin polypeptide from the corresponding wild-type mRNA. In someembodiments, the mRNA comprises an open ORF wherein uracil in the mRNAis at least about 95% 5-methoxyuracil, and wherein the uracil content ofthe ORF is between about 115% and about 135% of the theoretical minimumuracil content in the corresponding wild-type ORF, and wherein the mRNAdoes not substantially induce an innate immune response of a mammaliancell into which the mRNA is introduced.

10. Methods for Modifying Polynucleotides

The invention includes modified polynucleotides comprising apolynucleotide described herein (e.g., a polynucleotide, e.g., an mRNA,comprising a nucleotide sequence encoding a Citrin polypeptide). Themodified polynucleotides can be chemically modified and/or structurallymodified. When the polynucleotides of the present invention arechemically and/or structurally modified the polynucleotides can bereferred to as “modified polynucleotides.”

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides) encoding a Citrin polypeptide. A “nucleoside” refers toa compound containing a sugar molecule (e.g., a pentose or ribose) or aderivative thereof in combination with an organic base (e.g., a purineor pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). A “nucleotide” refers to a nucleoside including aphosphate group. Modified nucleotides can by synthesized by any usefulmethod, such as, for example, chemically, enzymatically, orrecombinantly, to include one or more modified or non-naturalnucleosides. Polynucleotides can comprise a region or regions of linkednucleosides. Such regions can have variable backbone linkages. Thelinkages can be standard phosphodiester linkages, in which case thepolynucleotides would comprise regions of nucleotides.

The modified polynucleotides disclosed herein can comprise variousdistinct modifications. In some embodiments, the modifiedpolynucleotides contain one, two, or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedpolynucleotide, introduced to a cell can exhibit one or more desirableproperties, e.g., improved protein expression, reduced immunogenicity,or reduced degradation in the cell, as compared to an unmodifiedpolynucleotide.

a. Structural Modifications

In some embodiments, a polynucleotide of the present invention (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) is structurally modified. As used herein, a “structural”modification is one in which two or more linked nucleosides areinserted, deleted, duplicated, inverted or randomized in apolynucleotide without significant chemical modification to thenucleotides themselves. Because chemical bonds will necessarily bebroken and reformed to effect a structural modification, structuralmodifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” can be chemically modified to “AT-5meC-G”. The samepolynucleotide can be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

b. Chemical Modifications

In some embodiments, the polynucleotides of the present invention arechemically modified. As used herein in reference to a polynucleotide,the terms “chemical modification” or, as appropriate, “chemicallymodified” refer to modification with respect to adenosine (A), guanosine(G), uridine (U), thymidine (T) or cytidine (C) ribo- ordeoxyribonucleosides in one or more of their position, pattern, percentor population. Generally, herein, these terms are not intended to referto the ribonucleotide modifications in naturally occurring 5′-terminalmRNA cap moieties.

In some embodiments, the polynucleotides of the present invention canhave a uniform chemical modification of all or any of the samenucleoside type or a population of modifications produced by meredownward titration of the same starting modification in all or any ofthe same nucleoside type, or a measured percent of a chemicalmodification of all any of the same nucleoside type but with randomincorporation, such as where all uridines are replaced by a uridineanalog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment,the polynucleotides can have a uniform chemical modification of two,three, or four of the same nucleoside type throughout the entirepolynucleotide (such as all uridines and all cytosines, etc. aremodified in the same way).

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil. Any combination ofbase/sugar or linker can be incorporated into polynucleotides of thepresent disclosure.

The skilled artisan will appreciate that, except where otherwise noted,polynucleotide sequences set forth in the instant application willrecite “T”s in a representative DNA sequence but where the sequencerepresents RNA, the “T”s would be substituted for “U”s.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) that are useful in the compositions, methods andsynthetic processes of the present disclosure include, but are notlimited to the following nucleotides, nucleosides, and nucleobases:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine;N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine;1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine;2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate);Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6, N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-α-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo) cytosine; 5 (methyl) cytosine; 5 (propynyl) cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyosine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-ethyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methyluridine,), 5-methoxyuridine; 5-methyl-2-thiouridine;5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; uridine 5-oxyaceticacid; uridine 5-oxyacetic acid methyl ester;3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2′ deoxy uridine; 2′fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2′ methyl,2′amino, 2′azido, 2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP;2′-Azido-2′-deoxy-UTP; 2′-Azido-deoxyuridine TP;2′-O-methylpseudouridine; 2′ deoxy uridine; 2′ fluorouridine;2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridine TP;2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP; Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy]-ethoxy)-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxyl}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; O6-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) includes a combination of at least two (e.g., 2,3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the mRNA comprises at least one chemically modifiednucleoside. In some embodiments, the at least one chemically modifiednucleoside is selected from the group consisting of pseudouridine (ψ),2-thiouridine (s2U), 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2′-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ),5-methoxy-uridine (moSU), 5-methyl-cytidine (m5C), α-thio-guanosine,α-thio-adenosine, 5-cyano uridine, 4′-thio uridine 7-deaza-adenine,1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine(m6A), and 2,6-Diaminopurine, (I), 1-methyl-inosine (m1I), wyosine(imG), methylwyosine (mimG), 7-deaza-guanosine,7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine(preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G),8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine,2-geranylthiouridine, 2-lysidine, 2-selenouridine,3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine,3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine,5-(carboxyhydroxymethyl)-2′-O-methyluridine methyl ester,5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine,5-aminomethyluridine, 5-carbamoylhydroxymethyluridine,5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine,5-carboxymethylaminomethyl-2-geranylthiouridine,5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine,5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine,7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine,7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine,N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine,agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-queuosine,methylated undermodified hydroxywybutosine,N4,N4,2′-O-trimethylcytidine, geranylated5-methylaminomethyl-2-thiouridine, geranylated5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQ0base, preQ1base,and two or more combinations thereof. In some embodiments, the at leastone chemically modified nucleoside is selected from the group consistingof pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine,5-methylcytosine, 5-methoxyuridine, and a combination thereof. In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) includes a combination of at least two (e.g., 2, 3, 4 ormore) of the aforementioned modified nucleobases.

(i) Base Modifications

In certain embodiments, the chemical modification is at nucleobases inthe polynucleotides (e.g., RNA polynucleotide, such as mRNApolynucleotide). In some embodiments, modified nucleobases in thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)are selected from the group consisting of 1-methyl-pseudouridine (m1ψ),1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine(m5C), pseudouridine (ψ), α-thio-guanosine and α-thio-adenosine. In someembodiments, the polynucleotide includes a combination of at least two(e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) comprises pseudouridine (ψ) and5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprises1-methyl-pseudouridine (m1ψ). In some embodiments, the polynucleotide(e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises1-ethyl-pseudouridine (e1ψ). In some embodiments, the polynucleotide(e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises 1-ethyl-pseudouridine (e1ψ) and5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine(s2U). In some embodiments, the polynucleotide (e.g., RNApolynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprisesmethoxy-uridine (mo5U). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprises5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises 2′-O-methyl uridine. In some embodiments, thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)comprises 2′-O-methyl uridine and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises N6-methyl-adenosine (m6A). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises N6-methyl-adenosine (m6A) and5-methyl-cytidine (m5C).

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) is uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNAsequence are replaced with 5-methyl-cytidine (m5C). Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as any of those set forth above.

In some embodiments, the chemically modified nucleosides in the openreading frame are selected from the group consisting of uridine,adenine, cytosine, guanine, and any combination thereof.

In some embodiments, the modified nucleobase is a modified cytosine.Examples of nucleobases and nucleosides having a modified cytosineinclude N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C),5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine(hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Example nucleobases and nucleosides having a modified uridine include5-cyano uridine or 4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Example nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A),N6-methyl-adenine (m6A), and 2,6-Diaminopurine.

In some embodiments, a modified nucleobase is a modified guanine.Example nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

In some embodiments, the nucleobase modified nucleotides in thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)are 5-methoxyuridine.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) includes a combination of at least two (e.g., 2,3, 4 or more) of modified nucleobases.

In some embodiments, at least 95% of a type of nucleobases (e.g.,uracil) in a polynucleotide of the invention (e.g., an mRNA encodingCitrin) are modified nucleobases. In some embodiments, at least 95% ofuracil in a polynucleotide of the present invention (e.g., an mRNAencoding Citrin) is 5-methoxyuracil.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) comprises 5-methoxyuridine (5mo5U) and5-methyl-cytidine (m5C).

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) is uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methoxyuridine, meaning that substantially all uridine residues in themRNA sequence are replaced with 5-methoxyuridine. Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as any of those set forth above.

In some embodiments, the modified nucleobase is a modified cytosine.

In some embodiments, a modified nucleobase is a modified uracil. Examplenucleobases and nucleosides having a modified uracil include5-methoxyuracil.

In some embodiments, a modified nucleobase is a modified adenine.

In some embodiments, a modified nucleobase is a modified guanine.

In some embodiments, the nucleobases, sugar, backbone, or anycombination thereof in the open reading frame encoding a Citrinpolypeptide are chemically modified by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, or 100%.

In some embodiments, the uridine nucleosides in the open reading frameencoding a Citrin polypeptide, e.g., a Citrin polypeptide or afunctional fragment or variant thereof, are chemically modified by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%.

In some embodiments, the adenosine nucleosides in the open reading frameencoding a Citrin polypeptide are chemically modified by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%.

In some embodiments, the cytidine nucleosides in the open reading frameencoding a Citrin polypeptide are chemically modified by at least atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%.

In some embodiments, the guanosine nucleosides in the open reading frameencoding a Citrin polypeptide are chemically modified by at least atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%.

In some embodiments, the polynucleotides can include any useful linkerbetween the nucleosides. Such linkers, including backbone modifications,that are useful in the composition of the present disclosure include,but are not limited to the following: 3′-alkylene phosphonates, 3′-aminophosphoramidate, alkene containing backbones,aminoalkylphosphoramidates, aminoalkylphosphotriesters,boranophosphates, —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—,—CH₂—NH—CH₂—, chiral phosphonates, chiral phosphorothioates, formacetyland thioformacetyl backbones, methylene (methylimino), methyleneformacetyl and thioformacetyl backbones, methyleneimino andmethylenehydrazino backbones, morpholino linkages, —N(CH₃)—CH₂—CH₂—,oligonucleosides with heteroatom internucleoside linkage, phosphinates,phosphoramidates, phosphorodithioates, phosphorothioate internucleosidelinkages, phosphorothioates, phosphotriesters, PNA, siloxane backbones,sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonateand sulfonamide backbones, thionoalkylphosphonates,thionoalkylphosphotriesters, and thionophosphoramidates.

(ii) Sugar Modifications

The modified nucleosides and nucleotides (e.g., building blockmolecules), which can be incorporated into a polynucleotide (e.g., RNAor mRNA, as described herein), can be modified on the sugar of theribonucleic acid. For example, the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different substituents. Exemplarysubstitutions at the 2′-position include, but are not limited to, H,halo, optionally substituted C₁₋₆ alkyl; optionally substituted C₁₋₆alkoxy; optionally substituted C₆₋₁₀ aryloxy; optionally substitutedC₃₋₈ cycloalkyl; optionally substituted C₃₋₈ cycloalkoxy; optionallysubstituted C₆₋₁₀ aryloxy; optionally substituted C₆₋₁₀ aryl-C₁₋₆alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy; a sugar (e.g.,ribose, pentose, or any described herein); a polyethyleneglycol (PEG),—O(CH₂CH₂O)_(n)CH₂CH₂OR, where R is H or optionally substituted alkyl,and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16,from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to20); “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connectedby a C₁₋₆ alkylene or C₁₋₆ heteroalkylene bridge to the 4′-carbon of thesame ribose sugar, where exemplary bridges included methylene,propylene, ether, or amino bridges; aminoalkyl, as defined herein;aminoalkoxy, as defined herein; amino as defined herein; and amino acid,as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide moleculecan include nucleotides containing, e.g., arabinose, as the sugar. Suchsugar modifications are taught International Patent Publication Nos.WO2013052523 and WO2014093924, the contents of each of which areincorporated herein by reference in their entireties.

(iii) Combinations of Modifications

The polynucleotides of the invention (e.g., a polynucleotide comprisinga nucleotide sequence encoding a Citrin polypeptide or a functionalfragment or variant thereof) can include a combination of modificationsto the sugar, the nucleobase, and/or the internucleoside linkage. Thesecombinations can include any one or more modifications described herein.

Combinations of modified nucleotides can be used to form thepolynucleotides of the invention. Unless otherwise noted, the modifiednucleotides can be completely substituted for the natural nucleotides ofthe polynucleotides of the invention. As a non-limiting example, thenatural nucleotide uridine can be substituted with a modified nucleosidedescribed herein. In another non-limiting example, the naturalnucleotide uridine can be partially substituted or replaced (e.g., about0%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 99%) with at least one of the modifiednucleoside disclosed herein. Any combination of base/sugar or linker canbe incorporated into the polynucleotides of the invention and suchmodifications are taught in International Patent PublicationsWO2013052523 and WO2014093924, and U.S. Publ. Nos. US 20130115272 andUS20150307542, the contents of each of which are incorporated herein byreference in its entirety.

11. Untranslated Regions (UTRs)

Untranslated regions (UTRs) are nucleic acid sections of apolynucleotide before a start codon (5′UTR) and after a stop codon(3′UTR) that are not translated. In some embodiments, a polynucleotide(e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of theinvention comprising an open reading frame (ORF) encoding a Citrinpolypeptide further comprises UTR (e.g., a 5′UTR or functional fragmentthereof, a 3′UTR or functional fragment thereof, or a combinationthereof).

A UTR can be homologous or heterologous to the coding region in apolynucleotide. In some embodiments, the UTR is homologous to the ORFencoding the Citrin polypeptide. In some embodiments, the UTR isheterologous to the ORF encoding the Citrin polypeptide. In someembodiments, the polynucleotide comprises two or more 5′UTRs orfunctional fragments thereof, each of which has the same or differentnucleotide sequences. In some embodiments, the polynucleotide comprisestwo or more 3′UTRs or functional fragments thereof, each of which hasthe same or different nucleotide sequences.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR orfunctional fragment thereof, or any combination thereof is sequenceoptimized.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR orfunctional fragment thereof, or any combination thereof comprises atleast one chemically modified nucleobase, e.g., 1-methylpseudouridine or5-methoxyuracil.

UTRs can have features that provide a regulatory role, e.g., increasedor decreased stability, localization and/or translation efficiency. Apolynucleotide comprising a UTR can be administered to a cell, tissue,or organism, and one or more regulatory features can be measured usingroutine methods. In some embodiments, a functional fragment of a 5′UTRor 3′UTR comprises one or more regulatory features of a full length 5′or 3′ UTR, respectively.

Natural 5′UTRs bear features that play roles in translation initiation.They harbor signatures like Kozak sequences that are commonly known tobe involved in the process by which the ribosome initiates translationof many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, whereR is a purine (adenine or guanine) three bases upstream of the startcodon (AUG), which is followed by another ‘G’. 5′UTRs also have beenknown to form secondary structures that are involved in elongationfactor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of a polynucleotide. For example, introduction of5′UTR of liver-expressed mRNA, such as albumin, serum amyloid A,Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, orFactor VIII, can enhance expression of polynucleotides in hepatic celllines or liver. Likewise, use of 5′UTR from other tissue-specific mRNAto improve expression in that tissue is possible for muscle (e.g., MyoD,Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g.,Tie-1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF,CD11b, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adiposetissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelialcells (e.g., SP-A/B/C/D).

In some embodiments, UTRs are selected from a family of transcriptswhose proteins share a common function, structure, feature or property.For example, an encoded polypeptide can belong to a family of proteins(i.e., that share at least one function, structure, feature,localization, origin, or expression pattern), which are expressed in aparticular cell, tissue or at some time during development. The UTRsfrom any of the genes or mRNA can be swapped for any other UTR of thesame or different family of proteins to create a new polynucleotide.

In some embodiments, the 5′UTR and the 3′UTR can be heterologous. Insome embodiments, the 5′UTR can be derived from a different species thanthe 3′UTR. In some embodiments, the 3′UTR can be derived from adifferent species than the 5′UTR.

Co-owned International Patent Application No. PCT/US2014/021522 (Publ.No. WO/2014/164253, incorporated herein by reference in its entirety)provides a listing of exemplary UTRs that can be utilized in thepolynucleotide of the present invention as flanking regions to an ORF.

Exemplary UTRs of the application include, but are not limited to, oneor more 5′UTR and/or 3′UTR derived from the nucleic acid sequence of: aglobin, such as an α- or β-globin (e.g., a Xenopus, mouse, rabbit, orhuman globin); a strong Kozak translational initiation signal; a CYBA(e.g., human cytochrome b-245 α polypeptide); an albumin (e.g., humanalbumin7); a HSD17B4 (hydroxysteroid (17-β) dehydrogenase); a virus(e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitisvirus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMVimmediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), asindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein(e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucosetransporter (e.g., hGLUT1 (human glucose transporter 1)); an actin(e.g., human a or actin); a GAPDH; a tubulin; a histone; a citric acidcycle enzyme; a topoisomerase (e.g., a 5′UTR of a TOP gene lacking the5′ TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32(L32); a ribosomal protein (e.g., human or mouse ribosomal protein, suchas, for example, rps9); an ATP synthase (e.g., ATP5A1 or the β subunitof mitochondrial H⁺-ATP synthase); a growth hormone e (e.g., bovine(bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1α1 (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyteenhancer factor 2A (MEF2A); a β-F1-ATPase, a creatine kinase, amyoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen(e.g., collagen type I, alpha 2 (Col1A2), collagen type I, alpha 1(Col1A1), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1(Col6A1)); a ribophorin (e.g., ribophorin I (RPNI)); a low densitylipoprotein receptor-related protein (e.g., LRP1); a cardiotrophin-likecytokine factor (e.g., Nnt1); calreticulin (Calr); a procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (Plod1); and a nucleobindin (e.g.,Nucb1).

In some embodiments, the 5′UTR is selected from the group consisting ofa β-globin 5′UTR; a 5′UTR containing a strong Kozak translationalinitiation signal; a cytochrome b-245 α polypeptide (CYBA) 5′UTR; ahydroxysteroid (17-β) dehydrogenase (HSD17B4) 5′UTR; a Tobacco etchvirus (TEV) 5′UTR; a Venezuelen equine encephalitis virus (TEEV) 5′UTR;a 5′ proximal open reading frame of rubella virus (RV) RNA encodingnonstructural proteins; a Dengue virus (DEN) 5′UTR; a heat shock protein70 (Hsp70) 5′UTR; a eIF4G 5′UTR; a GLUT1 5′UTR; functional fragmentsthereof and any combination thereof.

In some embodiments, the 3′UTR is selected from the group consisting ofa β-globin 3′UTR; a CYBA 3′UTR; an albumin 3′UTR; a growth hormone (GH)3′UTR; a VEEV 3′UTR; a hepatitis B virus (HBV) 3′UTR; α-globin 3′UTR; aDEN 3′UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3′UTR; anelongation factor 1 al (EEF1A1) 3′UTR; a manganese superoxide dismutase(MnSOD) 3′UTR; a β subunit of mitochondrial H(+)-ATP synthase (β-mRNA)3′UTR; a GLUT1 3′UTR; a MEF2A 3′UTR; a β-F1-ATPase 3′UTR; functionalfragments thereof and combinations thereof.

Wild-type UTRs derived from any gene or mRNA can be incorporated intothe polynucleotides of the invention. In some embodiments, a UTR can bealtered relative to a wild type or native UTR to produce a variant UTR,e.g., by changing the orientation or location of the UTR relative to theORF; or by inclusion of additional nucleotides, deletion of nucleotides,swapping or transposition of nucleotides. In some embodiments, variantsof 5′ or 3′ UTRs can be utilized, for example, mutants of wild typeUTRs, or variants wherein one or more nucleotides are added to orremoved from a terminus of the UTR.

Additionally, one or more synthetic UTRs can be used in combination withone or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat.Protoc. 2013 8(3):568-82, and sequences available atwww.addgene.org/Derrick_Rossi/, the contents of each are incorporatedherein by reference in their entirety. UTRs or portions thereof can beplaced in the same orientation as in the transcript from which they wereselected or can be altered in orientation or location. Hence, a 5′and/or 3′ UTR can be inverted, shortened, lengthened, or combined withone or more other 5′ UTRs or 3′ UTRs.

In some embodiments, the polynucleotide comprises multiple UTRs, e.g., adouble, a triple or a quadruple 5′UTR or 3′UTR. For example, a doubleUTR comprises two copies of the same UTR either in series orsubstantially in series. For example, a double beta-globin 3′UTR can beused (see US2010/0129877, the contents of which are incorporated hereinby reference in its entirety).

In certain embodiments, the polynucleotides of the invention comprise a5′UTR and/or a 3′UTR selected from any of the UTRs disclosed herein. Insome embodiments, the 5′UTR comprises:

5′UTR-001 (Upstream UTR) (SEQ ID NO. 30)(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′UTR-002 (Upstream UTR) (SEQ ID NO. 31)(GGGAGAU CAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′UTR-003 (Upstream UTR) (SEQ ID NO. 32); 5′UTR-004 (Upstream UTR)(SEQ ID NO. 33) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC);5′UTR-005 (Upstream UTR) (SEQ ID NO. 34)(GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′UTR-006 (Upstream UTR)(SEQ ID NO. 35); 5′UTR-007 (Upstream UTR)(SEQ ID NO. 36) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC);5′UTR-008 (Upstream UTR) (SEQ ID NO. 37)(GGGAAUUAACAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′UTR-009 (Upstream UTR) (SEQ ID NO. 38)(GGGAAAUUAGACAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);UTR 5′UTR-010, Upstream (SEQ ID NO. 39)(GGGAAAUAAGAGAGUAAAGAACAGUAAGAAGAAAUAUAAGAGCCACC);5′UTR-011 (Upstream UTR) (SEQ ID NO. 40)(GGGAAAAAAGAGAGAAAAGAAGACUAAGAAGAAAUAUAAGAGCCACC);5′UTR-012 (Upstream UTR) (SEQ ID NO. 41)(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAUAUAUAAGAGCCACC);5′UTR-013 (Upstream UTR) (SEQ ID NO. 42)(GGGAAAUAAGAGACAAAACAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′UTR-014 (Upstream UTR) (SEQ ID NO. 43)(GGGAAAUUAGAGAGUAAAGAACAGUAAGUAGAAUUAAAAGAGCCACC);5′UTR-15 (Upstream UTR) (SEQ ID NO. 44)(GGGAAAUAAGAGAGAAUAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′UTR-016 (Upstream UTR) (SEQ ID NO. 45)(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAAUUAAGAGCCACC);5′UTR-017 (Upstream UTR) (SEQ ID NO. 46); (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUUUAAGAGCCACC) or5′UTR-018 (Upstream UTR) (SEQ ID NO. 47)(UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC).

In some embodiments, the 3′UTR comprises:

142-3p 3 ′UTR (UTR including miR142-3p  binding site) (SEQ ID NO. 48)(UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC);142-3p 3 ′UTR (UTR including miR142-3p  binding site) (SEQ ID NO. 49)(UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC);142-3p 3 ′UTR (UTR including miR142-3p binding site) (SEQ ID NO. 50)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAAAGUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC);142-3p 3′UTR (UTR including miR142-3p binding site) (SEQ ID NO. 51)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC);142-3p 3′UTR (UTR including miR142-3p binding site) (SEQ ID NO. 52)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC);142-3p 3′UTR (UTR including miR142-3p binding site) (SEQ ID NO. 53)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC);142-3p 3′UTR (UTR including miR142-3p binding site) (SEQ ID NO. 54)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC);3′UTR-001 (Creatine Kinase UTR)  (See SEQ ID NO. 55);3′UTR-002 (Myoglobin UTR) (See SEQ ID NO. 56);3′UTR-003 (α-actin UTR) (See SEQ ID NO. 57);3′UTR-004 (Albumin UTR) (See SEQ ID NO. 58);3′UTR-005 (α-globin UTR) (See SEQ ID NO. 59);3′UTR-006 (G-CSF UTR) (See SEQ ID NO. 60);3′UTR-007 (Col1a2; collagen, type I, alpha 2 UTR) (See SEQ ID NO. 61);3′UTR-008 (Col6a2; collagen, type VI, alpha 2 UTR) (See SEQ ID NO. 62);3′UTR-009 (RPN1; ribophorin I UTR) (See SEQ ID NO. 63);3′UTR-010 (LRP1; low density lipoprotein receptor-related protein 1 UTR) (See SEQ ID NO. 64);3′UTR-011 (Nnt1; cardiotrophin-like cytokinefactor 1 UTR) (See SEQ ID NO. 65);3′UTR-012 (Col6a1; collagen, type VI, alpha 1 UTR) (See SEQ ID NO. 66);3′UTR-013 (Calr; calreticulin UTR)  (See SEQ ID NO. 67);3′UTR-014 (Col1a1; collagen, type I, alpha 1 UTR)  (See SEQ ID NO. 68);3′UTR-015 (Plodl; procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 UTR)  (See SEQ ID NO. 69);3′UTR-016 (Nucbl; nucleobindin 1 UTR)  (See SEQ ID NO. 70);3′UTR-017 (α-globin) (See SEQ ID NO. 71); 3′UTR-018 (See SEQ ID NO. 72);3′UTR (miR142 + 30miR126 binding sites variant 1) (SEQ ID NO. 105)UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC;3′UTR (miR142 + 30miR126 binding sites variant 2) (SEQ ID NO. 147)UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC; or3′UTR (miR142 binding site) (SEQ ID NO. 148)UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC.

In certain embodiments, the 5′UTR and/or 3′UTR sequence of the inventioncomprises a nucleotide sequence at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or about 100% identical to a sequence selected from the group consistingof 5′UTR sequences comprising any of SEQ ID NOs: 30-47, 79, 120-122,126-128 and/or 3′UTR sequences comprises any of SEQ ID NOs: 48-72, 80,81, 102-105, 108-117, 124, 125, 147-157, and any combination thereof.

The polynucleotides of the invention can comprise combinations offeatures. For example, the ORF can be flanked by a 5′UTR that comprisesa strong Kozak translational initiation signal and/or a 3′UTR comprisingan oligo(dT) sequence for templated addition of a poly-A tail. A 5′UTRcan comprise a first polynucleotide fragment and a second polynucleotidefragment from the same and/or different UTRs (see, e.g., US2010/0293625,herein incorporated by reference in its entirety).

Other non-UTR sequences can be used as regions or subregions within thepolynucleotides of the invention. For example, introns or portions ofintron sequences can be incorporated into the polynucleotides of theinvention. Incorporation of intronic sequences can increase proteinproduction as well as polynucleotide expression levels. In someembodiments, the polynucleotide of the invention comprises an internalribosome entry site (IRES) instead of or in addition to a UTR (see,e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010394(1):189-193, the contents of which are incorporated herein byreference in their entirety). In some embodiments, the polynucleotidecomprises an IRES instead of a 5′UTR sequence. In some embodiments, thepolynucleotide comprises an ORF and a viral capsid sequence. In someembodiments, the polynucleotide comprises a synthetic 5′UTR incombination with a non-synthetic 3′UTR.

In some embodiments, the UTR can also include at least one translationenhancer polynucleotide, translation enhancer element, or translationalenhancer elements (collectively, “TEE,” which refers to nucleic acidsequences that increase the amount of polypeptide or protein producedfrom a polynucleotide. As a non-limiting example, the TEE can includethose described in US2009/0226470, incorporated herein by reference inits entirety, and others known in the art. As a non-limiting example,the TEE can be located between the transcription promoter and the startcodon. In some embodiments, the 5′UTR comprises a TEE.

In one aspect, a TEE is a conserved element in a UTR that can promotetranslational activity of a nucleic acid such as, but not limited to,cap-dependent or cap-independent translation.

In one non-limiting example, the TEE comprises the TEE sequence in the5′-leader of the Gtx homeodomain protein. See Chappell et al., PNAS 2004101:9590-9594, incorporated herein by reference in its entirety.

In some embodiments, the polynucleotide of the invention comprises oneor multiple copies of a TEE. The TEE in a translational enhancerpolynucleotide can be organized in one or more sequence segments. Asequence segment can harbor one or more of the TEEs provided herein,with each TEE being present in one or more copies. When multiplesequence segments are present in a translational enhancerpolynucleotide, they can be homogenous or heterogeneous. Thus, themultiple sequence segments in a translational enhancer polynucleotidecan harbor identical or different types of the TEE provided herein,identical or different number of copies of each of the TEE, and/oridentical or different organization of the TEE within each sequencesegment. In one embodiment, the polynucleotide of the inventioncomprises a translational enhancer polynucleotide sequence. Non-limitingexamples of TEE sequences are described in U.S.

Publication 2014/0200261, the contents of which are incorporated hereinby reference in their entirety.

12. MicroRNA (miRNA) Binding Sites

Polynucleotides of the invention can include regulatory elements, forexample, microRNA (miRNA) binding sites, transcription factor bindingsites, structured mRNA sequences and/or motifs, artificial binding sitesengineered to act as pseudo-receptors for endogenous nucleic acidbinding molecules, and combinations thereof. In some embodiments,polynucleotides including such regulatory elements are referred to asincluding “sensor sequences”. Non-limiting examples of sensor sequencesare described in U.S. Publication 2014/0200261, the contents of whichare incorporated herein by reference in their entirety.

In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA),e.g., a messenger RNA (mRNA)) of the invention comprises an open readingframe (ORF) encoding a polypeptide of interest and further comprises oneor more miRNA binding site(s). Inclusion or incorporation of miRNAbinding site(s) provides for regulation of polynucleotides of theinvention, and in turn, of the polypeptides encoded therefrom, based ontissue-specific and/or cell-type specific expression ofnaturally-occurring miRNAs.

The present invention also provides pharmaceutical compositions andformulations that comprise any of the polynucleotides described above.In some embodiments, the composition or formulation further comprises adelivery agent.

In some embodiments, the composition or formulation can contain apolyribonucpolynucleotideleotide comprising a sequence optimized nucleicacid sequence disclosed herein which encodes a polypeptide. In someembodiments, the composition or formulation can contain a polynucleotide(e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF)having significant sequence identity to a sequence optimized nucleicacid sequence disclosed herein which encodes a polypeptide. In someembodiments, the polyribonucleotide further comprises a miRNA bindingsite, e.g., a miRNA binding site that binds miR-126, miR-142, miR-144,miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27 andmiR-26a.

A miRNA, e.g., a natural-occurring miRNA, is a 19-25 nucleotide longnoncoding RNA that binds to a polynucleotide and down-regulates geneexpression either by reducing stability or by inhibiting translation ofthe polynucleotide. A miRNA sequence comprises a “seed” region, i.e., asequence in the region of positions 2-8 of the mature miRNA. A miRNAseed can comprise positions 2-8 or 2-7 of the mature miRNA. In someembodiments, a miRNA seed can comprise 7 nucleotides (e.g., nucleotides2-8 of the mature miRNA), wherein the seed-complementary site in thecorresponding miRNA binding site is flanked by an adenosine (A) opposedto miRNA position 1. In some embodiments, a miRNA seed can comprise 6nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein theseed-complementary site in the corresponding miRNA binding site isflanked by an adenosine (A) opposed to miRNA position 1. See, forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. miRNA profiling of thetarget cells or tissues can be conducted to determine the presence orabsence of miRNA in the cells or tissues. In some embodiments, apolynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA(mRNA)) of the invention comprises one or more microRNA binding sites,microRNA target sequences, microRNA complementary sequences, or microRNAseed complementary sequences. Such sequences can correspond to, e.g.,have complementarity to, any known microRNA such as those taught in USPublication US2005/0261218 and US Publication US2005/0059005, thecontents of each of which are incorporated herein by reference in theirentirety.

microRNAs derive enzymatically from regions of RNA transcripts that foldback on themselves to form short hairpin structures often termed apre-miRNA (precursor-miRNA). A pre-miRNA typically has a two-nucleotideoverhang at its 3′ end, and has 3′ hydroxyl and 5′ phosphate groups.This precursor-mRNA is processed in the nucleus and subsequentlytransported to the cytoplasm where it is further processed by DICER (aRNase III enzyme), to form a mature microRNA of approximately 22nucleotides. The mature microRNA is then incorporated into a ribonuclearparticle to form the RNA-induced silencing complex, RISC, which mediatesgene silencing. Art-recognized nomenclature for mature miRNAs typicallydesignates the arm of the pre-miRNA from which the mature miRNA derives;“5p” means the microRNA is from the 5 prime arm of the pre-miRNA hairpinand “3p” means the microRNA is from the 3 prime end of the pre-miRNAhairpin. A miR referred to by number herein can refer to either of thetwo mature microRNAs originating from opposite arms of the samepre-miRNA (e.g., either the 3p or 5p microRNA). All miRs referred toherein are intended to include both the 3p and 5p arms/sequences, unlessparticularly specified by the 3p or 5p designation.

As used herein, the term “microRNA (miRNA or miR) binding site” refersto a sequence within a polynucleotide, e.g., within a DNA or within anRNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficientcomplementarity to all or a region of a miRNA to interact with,associate with or bind to the miRNA. In some embodiments, apolynucleotide of the invention comprising an ORF encoding a polypeptideof interest and further comprises one or more miRNA binding site(s). Inexemplary embodiments, a 5′UTR and/or 3′UTR of the polynucleotide (e.g.,a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprises theone or more miRNA binding site(s).

A miRNA binding site having sufficient complementarity to a miRNA refersto a degree of complementarity sufficient to facilitate miRNA-mediatedregulation of a polynucleotide, e.g., miRNA-mediated translationalrepression or degradation of the polynucleotide. In exemplary aspects ofthe invention, a miRNA binding site having sufficient complementarity tothe miRNA refers to a degree of complementarity sufficient to facilitatemiRNA-mediated degradation of the polynucleotide, e.g., miRNA-guidedRNA-induced silencing complex (RISC)-mediated cleavage of mRNA. ThemiRNA binding site can have complementarity to, for example, a 19-25nucleotide long miRNA sequence, to a 19-23 nucleotide long miRNAsequence, or to a 22 nucleotide long miRNA sequence. A miRNA bindingsite can be complementary to only a portion of a miRNA, e.g., to aportion less than 1, 2, 3, or 4 nucleotides of the full length of anaturally-occurring miRNA sequence, or to a portion less than 1, 2, 3,or 4 nucleotides shorter than a naturally-occurring miRNA sequence. Fullor complete complementarity (e.g., full complementarity or completecomplementarity over all or a significant portion of the length of anaturally-occurring miRNA) is preferred when the desired regulation ismRNA degradation.

In some embodiments, a miRNA binding site includes a sequence that hascomplementarity (e.g., partial or complete complementarity) with anmiRNA seed sequence. In some embodiments, the miRNA binding siteincludes a sequence that has complete complementarity with a miRNA seedsequence. In some embodiments, a miRNA binding site includes a sequencethat has complementarity (e.g., partial or complete complementarity)with an miRNA sequence. In some embodiments, the miRNA binding siteincludes a sequence that has complete complementarity with a miRNAsequence. In some embodiments, a miRNA binding site has completecomplementarity with a miRNA sequence but for 1, 2, or 3 nucleotidesubstitutions, terminal additions, and/or truncations.

In some embodiments, the miRNA binding site is the same length as thecorresponding miRNA. In other embodiments, the miRNA binding site isone, two, three, four, five, six, seven, eight, nine, ten, eleven ortwelve nucleotide(s) shorter than the corresponding miRNA at the 5′terminus, the 3′ terminus, or both. In still other embodiments, themicroRNA binding site is two nucleotides shorter than the correspondingmicroRNA at the 5′ terminus, the 3′ terminus, or both. The miRNA bindingsites that are shorter than the corresponding miRNAs are still capableof degrading the mRNA incorporating one or more of the miRNA bindingsites or preventing the mRNA from translation.

In some embodiments, the miRNA binding site binds the correspondingmature miRNA that is part of an active RISC containing Dicer. In anotherembodiment, binding of the miRNA binding site to the corresponding miRNAin RISC degrades the mRNA containing the miRNA binding site or preventsthe mRNA from being translated. In some embodiments, the miRNA bindingsite has sufficient complementarity to miRNA so that a RISC complexcomprising the miRNA cleaves the polynucleotide comprising the miRNAbinding site. In other embodiments, the miRNA binding site has imperfectcomplementarity so that a RISC complex comprising the miRNA inducesinstability in the polynucleotide comprising the miRNA binding site. Inanother embodiment, the miRNA binding site has imperfect complementarityso that a RISC complex comprising the miRNA represses transcription ofthe polynucleotide comprising the miRNA binding site.

In some embodiments, the miRNA binding site has one, two, three, four,five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) fromthe corresponding miRNA.

In some embodiments, the miRNA binding site has at least about ten, atleast about eleven, at least about twelve, at least about thirteen, atleast about fourteen, at least about fifteen, at least about sixteen, atleast about seventeen, at least about eighteen, at least about nineteen,at least about twenty, or at least about twenty-one contiguousnucleotides complementary to at least about ten, at least about eleven,at least about twelve, at least about thirteen, at least about fourteen,at least about fifteen, at least about sixteen, at least aboutseventeen, at least about eighteen, at least about nineteen, at leastabout twenty, or at least about twenty-one, respectively, contiguousnucleotides of the corresponding miRNA.

By engineering one or more miRNA binding sites into a polynucleotide ofthe invention, the polynucleotide can be targeted for degradation orreduced translation, provided the miRNA in question is available. Thiscan reduce off-target effects upon delivery of the polynucleotide. Forexample, if a polynucleotide of the invention is not intended to bedelivered to a tissue or cell but ends up is said tissue or cell, then amiRNA abundant in the tissue or cell can inhibit the expression of thegene of interest if one or multiple binding sites of the miRNA areengineered into the 5′UTR and/or 3′UTR of the polynucleotide. Thus, insome embodiments, incorporation of one or more miRNA binding sites intoan mRNA of the disclosure may reduce the hazard of off-target effectsupon nucleic acid molecule delivery and/or enable tissue-specificregulation of expression of a polypeptide encoded by the mRNA. In yetother embodiments, incorporation of one or more miRNA binding sites intoan mRNA of the disclosure can modulate immune responses upon nucleicacid delivery in vivo. In further embodiments, incorporation of one ormore miRNA binding sites into an mRNA of the disclosure can modulateaccelerated blood clearance (ABC) of lipid-comprising compounds andcompositions described herein.

Conversely, miRNA binding sites can be removed from polynucleotidesequences in which they naturally occur in order to increase proteinexpression in specific tissues. For example, a binding site for aspecific miRNA can be removed from a polynucleotide to improve proteinexpression in tissues or cells containing the miRNA.

Regulation of expression in multiple tissues can be accomplished throughintroduction or removal of one or more miRNA binding sites, e.g., one ormore distinct miRNA binding sites. The decision whether to remove orinsert a miRNA binding site can be made based on miRNA expressionpatterns and/or their profilings in tissues and/or cells in developmentand/or disease. Identification of miRNAs, miRNA binding sites, and theirexpression patterns and role in biology have been reported (e.g.,Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and ChereshCurr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 201226:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393-403 and all references therein;each of which is incorporated herein by reference in its entirety).

miRNAs and miRNA binding sites can correspond to any known sequence,including non-limiting examples described in U.S. Publication Nos.2014/0200261, 2005/0261218, and 2005/0059005, each of which areincorporated herein by reference in their entirety.

Examples of tissues where miRNA are known to regulate mRNA, and therebyprotein expression, include, but are not limited to, liver (miR-122),muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92,miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21,miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126).

Specifically, miRNAs are known to be differentially expressed in immunecells (also called hematopoietic cells), such as antigen presentingcells (APCs) (e.g., dendritic cells and macrophages), macrophages,monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killercells, etc. Immune cell specific miRNAs are involved in immunogenicity,autoimmunity, the immune-response to infection, inflammation, as well asunwanted immune response after gene therapy and tissue/organtransplantation. Immune cells specific miRNAs also regulate many aspectsof development, proliferation, differentiation and apoptosis ofhematopoietic cells (immune cells). For example, miR-142 and miR-146 areexclusively expressed in immune cells, particularly abundant in myeloiddendritic cells. It has been demonstrated that the immune response to apolynucleotide can be shut-off by adding miR-142 binding sites to the3′-UTR of the polynucleotide, enabling more stable gene transfer intissues and cells. miR-142 efficiently degrades exogenouspolynucleotides in antigen presenting cells and suppresses cytotoxicelimination of transduced cells (e.g., Annoni A et al., blood, 2009,114, 5152-5161; Brown B D, et al., Nat med. 2006, 12(5), 585-591; BrownB D, et al., blood, 2007, 110(13): 4144-4152, each of which isincorporated herein by reference in its entirety).

An antigen-mediated immune response can refer to an immune responsetriggered by foreign antigens, which, when entering an organism, areprocessed by the antigen presenting cells and displayed on the surfaceof the antigen presenting cells. T cells can recognize the presentedantigen and induce a cytotoxic elimination of cells that express theantigen.

Introducing a miR-142 binding site into the 5′UTR and/or 3′UTR of apolynucleotide of the invention can selectively repress gene expressionin antigen presenting cells through miR-142 mediated degradation,limiting antigen presentation in antigen presenting cells (e.g.,dendritic cells) and thereby preventing antigen-mediated immune responseafter the delivery of the polynucleotide. The polynucleotide is thenstably expressed in target tissues or cells without triggering cytotoxicelimination.

In one embodiment, binding sites for miRNAs that are known to beexpressed in immune cells, in particular, antigen presenting cells, canbe engineered into a polynucleotide of the invention to suppress theexpression of the polynucleotide in antigen presenting cells throughmiRNA mediated RNA degradation, subduing the antigen-mediated immuneresponse. Expression of the polynucleotide is maintained in non-immunecells where the immune cell specific miRNAs are not expressed. Forexample, in some embodiments, to prevent an immunogenic reaction againsta liver specific protein, any miR-122 binding site can be removed and amiR-142 (and/or mirR-146) binding site can be engineered into the 5′UTRand/or 3′UTR of a polynucleotide of the invention.

To further drive the selective degradation and suppression in APCs andmacrophage, a polynucleotide of the invention can include a furthernegative regulatory element in the 5′UTR and/or 3′UTR, either alone orin combination with miR-142 and/or miR-146 binding sites. As anon-limiting example, the further negative regulatory element is aConstitutive Decay Element (CDE).

Immune cell specific miRNAs include, but are not limited to,hsa-let-7α-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p,hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p,hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p,hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p,miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p,miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p,miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a-5p,miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p,miR-15a-3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p,miR-16-2-3p, miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p,miR-181a-2-3p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p,miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p, miR-27b-5p,miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-1-5p,miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p, miR-30e-5p,miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346,miR-34a-3p, miR-34a-5p, miR-363-3p, miR-363-5p, miR-372, miR-377-3p,miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p,miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935,miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR-99b-5p. Furthermore, novelmiRNAs can be identified in immune cell through micro-arrayhybridization and microtome analysis (e.g., Jima D D et al, Blood, 2010,116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, the content ofeach of which is incorporated herein by reference in its entirety.)

miRNAs that are known to be expressed in the liver include, but are notlimited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p,miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152,miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p,miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.MiRNA binding sites from any liver specific miRNA can be introduced toor removed from a polynucleotide of the invention to regulate expressionof the polynucleotide in the liver. Liver specific miRNA binding sitescan be engineered alone or further in combination with immune cell(e.g., APC) miRNA binding sites in a polynucleotide of the invention.

miRNAs that are known to be expressed in the lung include, but are notlimited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p,miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p,miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p,miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p,miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, andmiR-381-5p. miRNA binding sites from any lung specific miRNA can beintroduced to or removed from a polynucleotide of the invention toregulate expression of the polynucleotide in the lung. Lung specificmiRNA binding sites can be engineered alone or further in combinationwith immune cell (e.g., APC) miRNA binding sites in a polynucleotide ofthe invention.

miRNAs that are known to be expressed in the heart include, but are notlimited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p,miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p,miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p,miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p. mMiRNAbinding sites from any heart specific microRNA can be introduced to orremoved from a polynucleotide of the invention to regulate expression ofthe polynucleotide in the heart. Heart specific miRNA binding sites canbe engineered alone or further in combination with immune cell (e.g.,APC) miRNA binding sites in a polynucleotide of the invention.

miRNAs that are known to be expressed in the nervous system include, butare not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p,miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128,miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137,miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR-212-3p,miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p,miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329, miR-342-3p, miR-3665,miR-3666, miR-380-3p, miR-380-5p, miR-383, miR-410, miR-425-3p,miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510, miR-516a-3p,miR-548b-5p, miR-548c-5p, miR-571, miR-7-1-3p, miR-7-2-3p, miR-7-5p,miR-802, miR-922, miR-9-3p, and miR-9-5p. miRNAs enriched in the nervoussystem further include those specifically expressed in neurons,including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p,miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b,miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326,miR-328, miR-922 and those specifically expressed in glial cells,including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. miRNAbinding sites from any CNS specific miRNA can be introduced to orremoved from a polynucleotide of the invention to regulate expression ofthe polynucleotide in the nervous system. Nervous system specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of theinvention.

miRNAs that are known to be expressed in the pancreas include, but arenot limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p,miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p,miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p,miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944. MiRNA binding sitesfrom any pancreas specific miRNA can be introduced to or removed from apolynucleotide of the invention to regulate expression of thepolynucleotide in the pancreas. Pancreas specific miRNA binding sitescan be engineered alone or further in combination with immune cell (e.g.APC) miRNA binding sites in a polynucleotide of the invention.

miRNAs that are known to be expressed in the kidney include, but are notlimited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p,miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p,miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p,miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p,miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.miRNA binding sites from any kidney specific miRNA can be introduced toor removed from a polynucleotide of the invention to regulate expressionof the polynucleotide in the kidney. Kidney specific miRNA binding sitescan be engineered alone or further in combination with immune cell(e.g., APC) miRNA binding sites in a polynucleotide of the invention.

miRNAs that are known to be expressed in the muscle include, but are notlimited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b,miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p,miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p. MiRNAbinding sites from any muscle specific miRNA can be introduced to orremoved from a polynucleotide of the invention to regulate expression ofthe polynucleotide in the muscle. Muscle specific miRNA binding sitescan be engineered alone or further in combination with immune cell(e.g., APC) miRNA binding sites in a polynucleotide of the invention.

miRNAs are also differentially expressed in different types of cells,such as, but not limited to, endothelial cells, epithelial cells, andadipocytes.

miRNAs that are known to be expressed in endothelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p,miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p,miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p,miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p,miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p,miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p,miR-23a-5p, miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p,miR-424-5p, miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p,miR-92b-3p, and miR-92b-5p. Many novel miRNAs are discovered inendothelial cells from deep-sequencing analysis (e.g., Voellenkle C etal., RNA, 2012, 18, 472-484, herein incorporated by reference in itsentirety). miRNA binding sites from any endothelial cell specific miRNAcan be introduced to or removed from a polynucleotide of the inventionto regulate expression of the polynucleotide in the endothelial cells.

miRNAs that are known to be expressed in epithelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p,miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p,miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a,miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific inrespiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b,miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5pspecific in renal epithelial cells, and miR-762 specific in cornealepithelial cells. miRNA binding sites from any epithelial cell specificmiRNA can be introduced to or removed from a polynucleotide of theinvention to regulate expression of the polynucleotide in the epithelialcells.

In addition, a large group of miRNAs are enriched in embryonic stemcells, controlling stem cell self-renewal as well as the developmentand/or differentiation of various cell lineages, such as neural cells,cardiac, hematopoietic cells, skin cells, osteogenic cells and musclecells (e.g., Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764;Vidigal J A and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436;Goff L A et al., PLoS One, 2009, 4:e7192; Morin R D et al., Genome Res,2008,18, 610-621; Yoo J K et al., Stem Cells Dev. 2012, 21(11),2049-2057, each of which is herein incorporated by reference in itsentirety). MiRNAs abundant in embryonic stem cells include, but are notlimited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p,miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p,miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p,miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p,miR-423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f,miR-548g-3p, miR-548g-5p, miR-548i, miR-548k, miR-5481, miR-548m,miR-548n, miR-5480-3p, miR-5480-5p, miR-548p, miR-664a-3p, miR-664a-5p,miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p,miR-885-5p, miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p,miR-99b-3p and miR-99b-5p. Many predicted novel miRNAs are discovered bydeep sequencing in human embryonic stem cells (e.g., Morin R D et al.,Genome Res, 2008,18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each ofwhich is incorporated herein by reference in its entirety).

In one embodiment, the binding sites of embryonic stem cell specificmiRNAs can be included in or removed from the 3′UTR of a polynucleotideof the invention to modulate the development and/or differentiation ofembryonic stem cells, to inhibit the senescence of stem cells in adegenerative condition (e.g. degenerative diseases), or to stimulate thesenescence and apoptosis of stem cells in a disease condition (e.g.cancer stem cells).

In some embodiments, miRNAs are selected based on expression andabundance in immune cells of the hematopoietic lineage, such as B cells,T cells, macrophages, dendritic cells, and cells that are known toexpress TLR7/TLR8 and/or able to secrete cytokines such as endothelialcells and platelets. In some embodiments, the miRNA set thus includesmiRs that may be responsible in part for the immunogenicity of thesecells, and such that a corresponding miR-site incorporation inpolynucleotides of the present invention (e.g., mRNAs) could lead todestabilization of the mRNA and/or suppression of translation from thesemRNAs in the specific cell type. Non-limiting representative examplesinclude miR-142, miR-144, miR-150, miR-155 and miR-223, which arespecific for many of the hematopoietic cells; miR-142, miR150, miR-16and miR-223, which are expressed in B cells; miR-223, miR-451, miR-26a,miR-16, which are expressed in progenitor hematopoietic cells; andmiR-126, which is expressed in plasmacytoid dendritic cells, plateletsand endothelial cells. For further discussion of tissue expression ofmiRs see e.g., Teruel-Montoya, R. et al. (2014) PLoS One 9:e102259;Landgraf, P. et al. (2007) Cell 129:1401-1414; Bissels, U. et al. (2009)RNA 15:2375-2384. Any one miR-site incorporation in the 3′UTR and/or 5′UTR may mediate such effects in multiple cell types of interest (e.g.,miR-142 is abundant in both B cells and dendritic cells).

In some embodiments, it may be beneficial to target the same cell typewith multiple miRs and to incorporate binding sites to each of the 3pand 5p arm if both are abundant (e.g., both miR-142-3p and miR142-5p areabundant in hematopoietic stem cells). Thus, in certain embodiments,polynucleotides of the invention contain two or more (e.g., two, three,four or more) miR bindings sites from: (i) the group consisting ofmiR-142, miR-144, miR-150, miR-155 and miR-223 (which are expressed inmany hematopoietic cells); or (ii) the group consisting of miR-142,miR150, miR-16 and miR-223 (which are expressed in B cells); or thegroup consisting of miR-223, miR-451, miR-26a, miR-16 (which areexpressed in progenitor hematopoietic cells).

In some embodiments, it may also be beneficial to combine various miRssuch that multiple cell types of interest are targeted at the same time(e.g., miR-142 and miR-126 to target many cells of the hematopoieticlineage and endothelial cells). Thus, for example, in certainembodiments, polynucleotides of the invention comprise two or more(e.g., two, three, four or more) miRNA bindings sites, wherein: (i) atleast one of the miRs targets cells of the hematopoietic lineage (e.g.,miR-142, miR-144, miR-150, miR-155 or miR-223) and at least one of themiRs targets plasmacytoid dendritic cells, platelets or endothelialcells (e.g., miR-126); or (ii) at least one of the miRs targets B cells(e.g., miR-142, miR150, miR-16 or miR-223) and at least one of the miRstargets plasmacytoid dendritic cells, platelets or endothelial cells(e.g., miR-126); or (iii) at least one of the miRs targets progenitorhematopoietic cells (e.g., miR-223, miR-451, miR-26a or miR-16) and atleast one of the miRs targets plasmacytoid dendritic cells, platelets orendothelial cells (e.g., miR-126); or (iv) at least one of the miRstargets cells of the hematopoietic lineage (e.g., miR-142, miR-144,miR-150, miR-155 or miR-223), at least one of the miRs targets B cells(e.g., miR-142, miR150, miR-16 or miR-223) and at least one of the miRstargets plasmacytoid dendritic cells, platelets or endothelial cells(e.g., miR-126); or any other possible combination of the foregoing fourclasses of miR binding sites (i.e., those targeting the hematopoieticlineage, those targeting B cells, those targeting progenitorhematopoietic cells and/or those targeting plamacytoid dendriticcells/platelets/endothelial cells).

In one embodiment, to modulate immune responses, polynucleotides of thepresent invention can comprise one or more miRNA binding sequences thatbind to one or more miRs that are expressed in conventional immune cellsor any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatorycytokines and/or chemokines (e.g., in immune cells of peripherallymphoid organs and/or splenocytes and/or endothelial cells). It has nowbeen discovered that incorporation into an mRNA of one or more miRs thatare expressed in conventional immune cells or any cell that expressesTLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/orchemokines (e.g., in immune cells of peripheral lymphoid organs and/orsplenocytes and/or endothelial cells) reduces or inhibits immune cellactivation (e.g., B cell activation, as measured by frequency ofactivated B cells) and/or cytokine production (e.g., production of IL-6,IFN-γ and/or TNFα). Furthermore, it has now been discovered thatincorporation into an mRNA of one or more miRs that are expressed inconventional immune cells or any cell that expresses TLR7 and/or TLR8and secrete pro-inflammatory cytokines and/or chemokines (e.g., inimmune cells of peripheral lymphoid organs and/or splenocytes and/orendothelial cells) can reduce or inhibit an anti-drug antibody (ADA)response against a protein of interest encoded by the mRNA.

In another embodiment, to modulate accelerated blood clearance of anpolynucleotide delivered in a lipid-comprising compound or composition,polynucleotides of the invention can comprise one or more miR bindingsequences that bind to one or more miRNAs expressed in conventionalimmune cells or any cell that expresses TLR7 and/or TLR8 and secretepro-inflammatory cytokines and/or chemokines (e.g., in immune cells ofperipheral lymphoid organs and/or splenocytes and/or endothelial cells).It has now been discovered that incorporation into an mRNA of one ormore miR binding sites reduces or inhibits accelerated blood clearance(ABC) of the lipid-comprising compound or composition for use indelivering the mRNA. Furthermore, it has now been discovered thatincorporation of one or more miR binding sites into an mRNA reducesserum levels of anti-PEG anti-IgM (e.g., reduces or inhibits the acuteproduction of IgMs that recognize polyethylene glycol (PEG) by B cells)and/or reduces or inhibits proliferation and/or activation ofplasmacytoid dendritic cells following administration of alipid-comprising compound or composition comprising the mRNA.

In some embodiments, miR sequences may correspond to any known microRNAexpressed in immune cells, including but not limited to those taught inUS Publication US2005/0261218 and US Publication US2005/0059005, thecontents of which are incorporated herein by reference in theirentirety. Non-limiting examples of miRs expressed in immune cellsinclude those expressed in spleen cells, myeloid cells, dendritic cells,plasmacytoid dendritic cells, B cells, T cells and/or macrophages. Forexample, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24 andmiR-27 are expressed in myeloid cells, miR-155 is expressed in dendriticcells, B cells and T cells, miR-146 is upregulated in macrophages uponTLR stimulation and miR-126 is expressed in plasmacytoid dendriticcells. In certain embodiments, the miR(s) is expressed abundantly orpreferentially in immune cells. For example, miR-142 (miR-142-3p and/ormiR-142-5p), miR-126 (miR-126-3p and/or miR-126-5p), miR-146 (miR-146-3pand/or miR-146-5p) and miR-155 (miR-155-3p and/or miR155-5p) areexpressed abundantly in immune cells. These microRNA sequences are knownin the art and, thus, one of ordinary skill in the art can readilydesign binding sequences or target sequences to which these microRNAswill bind based upon Watson-Crick complementarity.

Accordingly, in various embodiments, polynucleotides of the presentinvention comprise at least one microRNA binding site for a miR selectedfrom the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16,miR-21, miR-223, miR-24 and miR-27. In another embodiment, the mRNAcomprises at least two miR binding sites for microRNAs expressed inimmune cells. In various embodiments, the polynucleotide of theinvention comprises 1-4, one, two, three or four miR binding sites formicroRNAs expressed in immune cells. In another embodiment, thepolynucleotide of the invention comprises three miR binding sites. ThesemiR binding sites can be for microRNAs selected from the groupconsisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21,miR-223, miR-24, miR-27, and combinations thereof. In one embodiment,the polynucleotide of the invention comprises two or more (e.g., two,three, four) copies of the same miR binding site expressed in immunecells, e.g., two or more copies of a miR binding site selected from thegroup of miRs consisting of miR-142, miR-146, miR-155, miR-126, miR-16,miR-21, miR-223, miR-24, miR-27.

In one embodiment, the polynucleotide of the invention comprises threecopies of the same miR binding site. In certain embodiments, use ofthree copies of the same miR binding site can exhibit beneficialproperties as compared to use of a single miR binding site. Non-limitingexamples of sequences for 3′ UTRs containing three miR bindings sitesare shown in SEQ ID NO: 108 (three miR-142-3p binding sites) and SEQ IDNO: 110 (three miR-142-5p binding sites).

In another embodiment, the polynucleotide of the invention comprises twoor more (e.g., two, three, four) copies of at least two different miRbinding sites expressed in immune cells. Non-limiting examples ofsequences of 3′ UTRs containing two or more different miR binding sitesare shown in SEQ ID NO: 105 (one miR-142-3p binding site and onemiR-126-3p binding site), SEQ ID NO: 111 (two miR-142-5p binding sitesand one miR-142-3p binding sites) and SEQ ID NO: 114 (two miR-155-5pbinding sites and one miR-142-3p binding sites).

In another embodiment, the polynucleotide of the invention comprises atleast two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-142-3p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-142-3p and miR-155 (miR-155-3p or miR-155-5p), miR-142-3p andmiR-146 (miR-146-3 or miR-146-5p), or miR-142-3p and miR-126 (miR-126-3por miR-126-5p).

In another embodiment, the polynucleotide of the invention comprises atleast two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-126-3p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-126-3p and miR-155 (miR-155-3p or miR-155-5p), miR-126-3p andmiR-146 (miR-146-3p or miR-146-5p), or miR-126-3p and miR-142(miR-142-3p or miR-142-5p).

In another embodiment, the polynucleotide of the invention comprises atleast two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-142-5p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-142-5p and miR-155 (miR-155-3p or miR-155-5p), miR-142-5p andmiR-146 (miR-146-3 or miR-146-5p), or miR-142-5p and miR-126 (miR-126-3por miR-126-5p).

In yet another embodiment, the polynucleotide of the invention comprisesat least two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-155-5p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-155-5p and miR-142 (miR-142-3p or miR-142-5p), miR-155-5p andmiR-146 (miR-146-3 or miR-146-5p), or miR-155-5p and miR-126 (miR-126-3por miR-126-5p).

miRNA can also regulate complex biological processes such asangiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the polynucleotides of the invention, miRNA bindingsites that are involved in such processes can be removed or introduced,in order to tailor the expression of the polynucleotides to biologicallyrelevant cell types or relevant biological processes. In this context,the polynucleotides of the invention are defined as auxotrophicpolynucleotides.

In some embodiments, a polynucleotide of the invention comprises a miRNAbinding site, wherein the miRNA binding site comprises one or morenucleotide sequences selected from TABLE 3 and TABLE 4, including one ormore copies of any one or more of the miRNA binding site sequences. Insome embodiments, a polynucleotide of the invention further comprises atleast one, two, three, four, five, six, seven, eight, nine, ten, or moreof the same or different miRNA binding sites selected from TABLE 3 andTABLE 4, including any combination thereof.

In some embodiments, the miRNA binding site binds to miR-142 or iscomplementary to miR-142. In some embodiments, the miR-142 comprises SEQID NO:73. In some embodiments, the miRNA binding site binds tomiR-142-3p or miR-142-5p. In some embodiments, the miR-142-3p bindingsite comprises SEQ ID NO:75. In some embodiments, the miR-142-5p bindingsite comprises SEQ ID NO:77. In some embodiments, the miRNA binding sitecomprises a nucleotide sequence at least 80%, at least 85%, at least90%, at least 95%, or 100% identical to SEQ ID NO:75 or SEQ ID NO:77.

In some embodiments, the miRNA binding site binds to miR-126 or iscomplementary to miR-126. In some embodiments, the miR-126 comprises SEQID NO: 142. In some embodiments, the miRNA binding site binds tomiR-126-3p or miR-126-5p. In some embodiments, the miR-126-3p bindingsite comprises SEQ ID NO: 144. In some embodiments, the miR-126-5pbinding site comprises SEQ ID NO: 146. In some embodiments, the miRNAbinding site comprises a nucleotide sequence at least 80%, at least 85%,at least 90%, at least 95%, or 100% identical to SEQ ID NO: 144 or SEQID NO: 146.

In one embodiment, the 3′ UTR comprises two miRNA binding sites, whereina first miRNA binding site binds to miR-142 and a second miRNA bindingsite binds to miR-126. In a specific embodiment, the 3′ UTR binding tomiR-142 and miR-126 comprises, consists, or consists essentially of thesequence of SEQ ID NO: 105 or 147.

TABLE 3  miR-142, miR-126, and miR-142 and miR-126  binding sites SEQ IDNO. Description Sequence  73 miR-142 GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUG GAGGGUGUAGUGUUUCCUACUUU AUGGAUGAGUGUACUGUG  74miR-142-3p UGUAGUGUUUCCUACUUUAUGGA  75 miR-142-3pUCCAUAAAGUAGGAAACACUACA binding site  76 miR-142-5pCAUAAAGUAGAAAGCACUACU  77 miR-142-5p AGUAGUGCUUUCUACUUUAUG binding site142 miR-126 CGCUGGCGACGGGACAUUAUUAC UUUUGGUACGCGCUGUGACACUUCAAACUCGUACCGUGAGUAAUAA UGCGCCGUCCACGGCA 143 miR-126-3pUCGUACCGUGAGUAAUAAUGCG 144 miR-126-3p CGCAUUAUUACUCACGGUACGAbinding site 145 miR-126-5p CAUUAUUACUUUUGGUACGCG 146 miR-126-5pCGCGUACCAAAAGUAAUAAUG binding site

In some embodiments, a miRNA binding site is inserted in thepolynucleotide of the invention in any position of the polynucleotide(e.g., the 5′UTR and/or 3′UTR). In some embodiments, the 5′UTR comprisesa miRNA binding site. In some embodiments, the 3′UTR comprises a miRNAbinding site. In some embodiments, the 5′UTR and the 3′UTR comprise amiRNA binding site. The insertion site in the polynucleotide can beanywhere in the polynucleotide as long as the insertion of the miRNAbinding site in the polynucleotide does not interfere with thetranslation of a functional polypeptide in the absence of thecorresponding miRNA; and in the presence of the miRNA, the insertion ofthe miRNA binding site in the polynucleotide and the binding of themiRNA binding site to the corresponding miRNA are capable of degradingthe polynucleotide or preventing the translation of the polynucleotide.

In some embodiments, a miRNA binding site is inserted in at least about30 nucleotides downstream from the stop codon of an ORF in apolynucleotide of the invention comprising the ORF. In some embodiments,a miRNA binding site is inserted in at least about 10 nucleotides, atleast about 15 nucleotides, at least about 20 nucleotides, at leastabout 25 nucleotides, at least about 30 nucleotides, at least about 35nucleotides, at least about 40 nucleotides, at least about 45nucleotides, at least about 50 nucleotides, at least about 55nucleotides, at least about 60 nucleotides, at least about 65nucleotides, at least about 70 nucleotides, at least about 75nucleotides, at least about 80 nucleotides, at least about 85nucleotides, at least about 90 nucleotides, at least about 95nucleotides, or at least about 100 nucleotides downstream from the stopcodon of an ORF in a polynucleotide of the invention. In someembodiments, a miRNA binding site is inserted in about 10 nucleotides toabout 100 nucleotides, about 20 nucleotides to about 90 nucleotides,about 30 nucleotides to about 80 nucleotides, about 40 nucleotides toabout 70 nucleotides, about 50 nucleotides to about 60 nucleotides,about 45 nucleotides to about 65 nucleotides downstream from the stopcodon of an ORF in a polynucleotide of the invention.

In some embodiments, a miRNA binding site is inserted within the 3′ UTRimmediately following the stop codon of the coding region within thepolynucleotide of the invention, e.g., mRNA. In some embodiments, ifthere are multiple copies of a stop codon in the construct, a miRNAbinding site is inserted immediately following the final stop codon. Insome embodiments, a miRNA binding site is inserted further downstream ofthe stop codon, in which case there are 3′ UTR bases between the stopcodon and the miR binding site(s). In some embodiments, threenon-limiting examples of possible insertion sites for a miR in a 3′ UTRare shown in SEQ ID NOs: 115, 116, and 117, which show a 3′ UTR sequencewith a miR-142-3p site inserted in one of three different possibleinsertion sites, respectively, within the 3′ UTR.

In some embodiments, one or more miRNA binding sites can be positionedwithin the 5′ UTR at one or more possible insertion sites. For example,three non-limiting examples of possible insertion sites for a miR in a5′ UTR are shown in SEQ ID NOs: 120, 121, and 122, which show a 5′ UTRsequence with a miR-142-3p site inserted into one of three differentpossible insertion sites, respectively, within the 5′ UTR.

In one embodiment, a codon optimized open reading frame encoding apolypeptide of interest comprises a stop codon and the at least onemicroRNA binding site is located within the 3′ UTR 1-100 nucleotidesafter the stop codon. In one embodiment, the codon optimized openreading frame encoding the polypeptide of interest comprises a stopcodon and the at least one microRNA binding site for a miR expressed inimmune cells is located within the 3′ UTR 30-50 nucleotides after thestop codon. In another embodiment, the codon optimized open readingframe encoding the polypeptide of interest comprises a stop codon andthe at least one microRNA binding site for a miR expressed in immunecells is located within the 3′ UTR at least 50 nucleotides after thestop codon. In other embodiments, the codon optimized open reading frameencoding the polypeptide of interest comprises a stop codon and the atleast one microRNA binding site for a miR expressed in immune cells islocated within the 3′ UTR immediately after the stop codon, or withinthe 3′ UTR 15-20 nucleotides after the stop codon or within the 3′ UTR70-80 nucleotides after the stop codon. In other embodiments, the 3′UTRcomprises more than one miRNA binding site (e.g., 2-4 miRNA bindingsites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or30-50 nucleotides in length) between each miRNA binding site. In anotherembodiment, the 3′ UTR comprises a spacer region between the end of themiRNA binding site(s) and the poly A tail nucleotides. For example, aspacer region of 10-100, 20-70 or 30-50 nucleotides in length can besituated between the end of the miRNA binding site(s) and the beginningof the poly A tail.

In one embodiment, a codon optimized open reading frame encoding apolypeptide of interest comprises a start codon and the at least onemicroRNA binding site is located within the 5′ UTR 1-100 nucleotidesbefore (upstream of) the start codon. In one embodiment, the codonoptimized open reading frame encoding the polypeptide of interestcomprises a start codon and the at least one microRNA binding site for amiR expressed in immune cells is located within the 5′ UTR 10-50nucleotides before (upstream of) the start codon. In another embodiment,the codon optimized open reading frame encoding the polypeptide ofinterest comprises a start codon and the at least one microRNA bindingsite for a miR expressed in immune cells is located within the 5′ UTR atleast 25 nucleotides before (upstream of) the start codon. In otherembodiments, the codon optimized open reading frame encoding thepolypeptide of interest comprises a start codon and the at least onemicroRNA binding site for a miR expressed in immune cells is locatedwithin the 5′ UTR immediately before the start codon, or within the 5′UTR 15-20 nucleotides before the start codon or within the 5′ UTR 70-80nucleotides before the start codon. In other embodiments, the 5′UTRcomprises more than one miRNA binding site (e.g., 2-4 miRNA bindingsites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or30-50 nucleotides in length) between each miRNA binding site.

In one embodiment, the 3′ UTR comprises more than one stop codon,wherein at least one miRNA binding site is positioned downstream of thestop codons. For example, a 3′ UTR can comprise 1, 2 or 3 stop codons.Non-limiting examples of triple stop codons that can be used include:UGAUAAUAG, UGAUAGUAA, UAAUGAUAG, UGAUAAUAA, UGAUAGUAG, UAAUGAUGA,UAAUAGUAG, UGAUGAUGA, UAAUAAUAA and UAGUAGUAG. Within a 3′ UTR, forexample, 1, 2, 3 or 4 miRNA binding sites, e.g., miR-142-3p bindingsites, can be positioned immediately adjacent to the stop codon(s) or atany number of nucleotides downstream of the final stop codon. When the3′ UTR comprises multiple miRNA binding sites, these binding sites canbe positioned directly next to each other in the construct (i.e., oneafter the other) or, alternatively, spacer nucleotides can be positionedbetween each binding site.

In one embodiment, the 3′ UTR comprises three stop codons with a singlemiR-142-3p binding site located downstream of the 3rd stop codon.Non-limiting examples of sequences of 3′ UTR having three stop codonsand a single miR-142-3p binding site located at different positionsdownstream of the final stop codon are shown in SEQ ID NOs: 103 and115-117.

TABLE 4 3′UTRs, miR sequences, and miR binding sites SEQ ID NO: Sequence 81 GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 142-3p binding site)  82UCCAUAAAGUAGGAAACACUACA (miR 142-3p binding site)  83UGUAGUGUUUCCUACUUUAUGGA (miR 142-3p sequence)  84 CAUAAAGUAGAAAGCACUACU(miR 142-5p sequence)  85 CCUCUGAAAUUCAGUUCUUCAG (miR 146-3p sequence) 86 UGAGAACUGAAUUCCAUGGGUU (miR 146-5p sequence)  87CUCCUACAUAUUAGCAUUAACA (miR 155-3p sequence)  88 UUAAUGCUAAUCGUGAUAGGGGU(miR 155-5p sequence)  89 UCGUACCGUGAGUAAUAAUGCG (miR 126-3p sequence) 90 CAUUAUUACUUUUGGUACGCG (miR 126-5p sequence)  91CCAGUAUUAACUGUGCUGCUGA (miR 16-3p sequence)  92 UAGCAGCACGUAAAUAUUGGCG(miR 16-5p sequence)  93 CAACACCAGUCGAUGGGCUGU (miR 21-3p sequence)  94UAGCUUAUCAGACUGAUGUUGA (miR 21-5p sequence)  95 UGUCAGUUUGUCAAAUACCCCA(miR 223-3p sequence)  96 CGUGUAUUUGACAAGCUGAGUU (miR 223-5p sequence) 97 UGGCUCAGUUCAGCAGGAACAG (miR 24-3p sequence)  98UGCCUACUGAGCUGAUAUCAGU (miR 24-5p sequence)  99 UUCACAGUGGCUAAGUUCCGC(miR 27-3p sequence) 100 AGGGCUUAGCUGCUUGUGAGCA (miR 27-5p sequence) 101CGCAUUAUUACUCACGGUACGA (miR 126-3p binding site) 102UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 126-3p binding site)  72UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGC(3′UTR, no miR binding sites) 103UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site) 104UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 126-3p binding site) 105UGAUAAUAG UCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG UACCCCC

GUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC(3′UTR with miR 142-3p and miR 126-3p binding sites) 106UUAAUGCUAAUUGUGAUAGGGGU (miR 155-5p sequence) 107ACCCCUAUCACAAUUAGCAUUAA (miR 155-5p binding site) 108 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with 3 miR 142-3p binding sites)109 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 142-5p binding site) 110UGAUAAUAG

GCUGGAGCCUCGGUGGCCAUGC UUCUUGCCCCUUGGGCC

UCCCCCCAGCCCCU CCUCCCCUUCCUGCACCCGUACCCCC

GUGGU CUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with 3 miR 142-5p binding sites)111 UGAUAAUAG

GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with 2 miR 142-5p binding sites and 1 miR 142-3p  binding site)112 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 155-5p binding site) 113 UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCACCCCUAUCACAAUUAGCAUUAAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with 3 miR 155-5p binding sites)114 UGAUAAUAG ACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with 2 miR 155-5p binding sites and 1 miR 142-3p  binding site)115 UGAUAAUAG UCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P1 insertion) 116UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P2 insertion)  50UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAAAGUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3 ′UTR including miR142-3p binding site)  51UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR including miR142-3p binding site)  52UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR including including miR142-3p binding site)  54UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC(3′UTR including including miR142-3p binding site) 117UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P3 insertion) 118AGUAGUGCUUUCUACUUUAUG (miR-142-5p binding site) 119GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (miR-142)  30GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (5′UTR) 120GGGAAAUAAGAGUCCAUAAAGUAGGAAACACUACAAGAAAAGAAGAGUAAGA AGAAAUAUAAGAGCCACC(5′UTR with miR142-3p binding site at position pl) 121GGGAAAUAAGAGAGAAAAGAAGAGUAAUCCAUAAAGUAGGAAACACUACAGA AGAAAUAUAAGAGCCACC(5′UTR with miR142-3p binding site at position p2) 122GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAUCCAUAAAGUAGG AAACACUACAGAGCCACC(5′UTR with miR142-3p binding site at position p3) 124 UGAUAAUAG

GCUGGAGCCUCGGUGGCCAUGC UUCUUGCCCCUUGGGCC

UCCCCCCAGCCCCU CUCCCCUUCCUGCACCCGUACCCCC

GUGGUC UUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with 3 miR 142-5p binding sites)125 UGAUAAUAG

GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with 2 miR 142-5p binding sites and 1 miR 142-3p binding site)149 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGC(3′UTR, no miR binding sites variant 2) 148UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site variant 2) 150UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 126-3p binding site variant 3) 147 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG UACCCCC

GUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC(3′UTR with miR 142-3p and miR 126-3p binding sites variant 2) 151UGAUAAUAG UCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with 3 miR 142-3p binding sites variant 2) 152 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P1 insertion variant 2) 153UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P2 insertion variant 2) 154UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P3 insertion variant 2) 155UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 155-5p binding site variant 2) 156 UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCACCCCUAUCACAAUUAGCAUUAAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with 3 miR 155-5p binding sites variant 2) 157 UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with 2 miR 155-5p binding sites and 1 miR 142-3p binding sitevariant 2) Stop codon = bold miR 142-3p binding site = underline miR126-3p binding site = bold underline miR 155-5p binding site= italicized miR 142-5p binding site = italicized and bold underline

In one embodiment, the polynucleotide of the invention comprises a 5′UTR, a codon optimized open reading frame encoding a polypeptide ofinterest, a 3′ UTR comprising the at least one miRNA binding site for amiR expressed in immune cells, and a 3′ tailing region of linkednucleosides. In various embodiments, the 3′ UTR comprises 1-4, at leasttwo, one, two, three or four miRNA binding sites for miRs expressed inimmune cells, preferably abundantly or preferentially expressed inimmune cells.

In one embodiment, the at least one miRNA expressed in immune cells is amiR-142-3p microRNA binding site. In one embodiment, the miR-142-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 82. Inone embodiment, the 3′ UTR of the mRNA comprising the miR-142-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 81.

In one embodiment, the at least one miRNA expressed in immune cells is amiR-126 microRNA binding site. In one embodiment, the miR-126 bindingsite is a miR-126-3p binding site. In one embodiment, the miR-126-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 144. Inone embodiment, the 3′ UTR of the mRNA of the invention comprising themiR-126-3p microRNA binding site comprises the sequence shown in SEQ IDNO: 102.

Non-limiting exemplary sequences for miRs to which a microRNA bindingsite(s) of the disclosure can bind include the following: miR-142-3p(SEQ ID NO: 83), miR-142-5p (SEQ ID NO: 84), miR-146-3p (SEQ ID NO: 85),miR-146-5p (SEQ ID NO: 86), miR-155-3p (SEQ ID NO: 87), miR-155-5p (SEQID NO: 88), miR-126-3p (SEQ ID NO: 89), miR-126-5p (SEQ ID NO: 90),miR-16-3p (SEQ ID NO: 91), miR-16-5p (SEQ ID NO: 92), miR-21-3p (SEQ IDNO: 93), miR-21-5p (SEQ ID NO: 94), miR-223-3p (SEQ ID NO: 95),miR-223-5p (SEQ ID NO: 96), miR-24-3p (SEQ ID NO: 97), miR-24-5p (SEQ IDNO: 98), miR-27-3p (SEQ ID NO: 99) and miR-27-5p (SEQ ID NO:100).

Other suitable miR sequences expressed in immune cells (e.g., abundantlyor preferentially expressed in immune cells) are known and available inthe art, for example at the University of Manchester's microRNAdatabase, miRBase. Sites that bind any of the aforementioned miRs can bedesigned based on Watson-Crick complementarity to the miR, typically100% complementarity to the miR, and inserted into an mRNA construct ofthe disclosure as described herein.

In another embodiment, a polynucleotide of the present invention (e.g.,and mRNA, e.g., the 3′ UTR thereof) can comprise at least one miRNAbinding site to thereby reduce or inhibit accelerated blood clearance,for example by reducing or inhibiting production of IgMs, e.g., againstPEG, by B cells and/or reducing or inhibiting proliferation and/oractivation of pDCs, and can comprise at least one miRNA binding site formodulating tissue expression of an encoded protein of interest.

miRNA gene regulation can be influenced by the sequence surrounding themiRNA such as, but not limited to, the species of the surroundingsequence, the type of sequence (e.g., heterologous, homologous,exogenous, endogenous, or artificial), regulatory elements in thesurrounding sequence and/or structural elements in the surroundingsequence. The miRNA can be influenced by the 5′UTR and/or 3′UTR. As anon-limiting example, a non-human 3′UTR can increase the regulatoryeffect of the miRNA sequence on the expression of a polypeptide ofinterest compared to a human 3′UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elementsof the 5′UTR can influence miRNA mediated gene regulation. One exampleof a regulatory element and/or structural element is a structured IRES(Internal Ribosome Entry Site) in the 5′UTR, which is necessary for thebinding of translational elongation factors to initiate proteintranslation. EIF4A2 binding to this secondarily structured element inthe 5′-UTR is necessary for miRNA mediated gene expression (Meijer H Aet al., Science, 2013, 340, 82-85, herein incorporated by reference inits entirety). The polynucleotides of the invention can further includethis structured 5′UTR in order to enhance microRNA mediated generegulation.

At least one miRNA binding site can be engineered into the 3′UTR of apolynucleotide of the invention. In this context, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, or more miRNA binding sitescan be engineered into a 3′UTR of a polynucleotide of the invention. Forexample, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 2, or 1 miRNA binding sites can be engineered into the 3′UTR of apolynucleotide of the invention. In one embodiment, miRNA binding sitesincorporated into a polynucleotide of the invention can be the same orcan be different miRNA sites. A combination of different miRNA bindingsites incorporated into a polynucleotide of the invention can includecombinations in which more than one copy of any of the different miRNAsites are incorporated. In another embodiment, miRNA binding sitesincorporated into a polynucleotide of the invention can target the sameor different tissues in the body. As a non-limiting example, through theintroduction of tissue-, cell-type-, or disease-specific miRNA bindingsites in the 3′-UTR of a polynucleotide of the invention, the degree ofexpression in specific cell types (e.g., myeloid cells, endothelialcells, etc.)) can be reduced.

In one embodiment, a miRNA binding site can be engineered near the 5′terminus of the 3′UTR, about halfway between the 5′ terminus and 3′terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR in apolynucleotide of the invention. As a non-limiting example, a miRNAbinding site can be engineered near the 5′ terminus of the 3′UTR andabout halfway between the 5′ terminus and 3′ terminus of the 3′UTR. Asanother non-limiting example, a miRNA binding site can be engineerednear the 3′ terminus of the 3′UTR and about halfway between the 5′terminus and 3′ terminus of the 3′UTR. As yet another non-limitingexample, a miRNA binding site can be engineered near the 5′ terminus ofthe 3′UTR and near the 3′ terminus of the 3′UTR.

In another embodiment, a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 miRNA binding sites. The miRNA binding sites can be complementaryto a miRNA, miRNA seed sequence, and/or miRNA sequences flanking theseed sequence.

In some embodiments, the expression of a polynucleotide of the inventioncan be controlled by incorporating at least one sensor sequence in thepolynucleotide and formulating the polynucleotide for administration. Asa non-limiting example, a polynucleotide of the invention can betargeted to a tissue or cell by incorporating a miRNA binding site andformulating the polynucleotide in a lipid nanoparticle comprising anionizable lipid, including any of the lipids described herein.

A polynucleotide of the invention can be engineered for more targetedexpression in specific tissues, cell types, or biological conditionsbased on the expression patterns of miRNAs in the different tissues,cell types, or biological conditions. Through introduction oftissue-specific miRNA binding sites, a polynucleotide of the inventioncan be designed for optimal protein expression in a tissue or cell, orin the context of a biological condition.

In some embodiments, a polynucleotide of the invention can be designedto incorporate miRNA binding sites that either have 100% identity toknown miRNA seed sequences or have less than 100% identity to miRNA seedsequences. In some embodiments, a polynucleotide of the invention can bedesigned to incorporate miRNA binding sites that have at least: 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity toknown miRNA seed sequences. The miRNA seed sequence can be partiallymutated to decrease miRNA binding affinity and as such result in reduceddown modulation of the polynucleotide. In essence, the degree of matchor mis-match between the miRNA binding site and the miRNA seed can actas a rheostat to more finely tune the ability of the miRNA to modulateprotein expression. In addition, mutation in the non-seed region of amiRNA binding site can also impact the ability of a miRNA to modulateprotein expression.

In one embodiment, a miRNA sequence can be incorporated into the loop ofa stem loop.

In another embodiment, a miRNA seed sequence can be incorporated in theloop of a stem loop and a miRNA binding site can be incorporated intothe 5′ or 3′ stem of the stem loop.

In one embodiment, a translation enhancer element (TEE) can beincorporated on the 5′end of the stem of a stem loop and a miRNA seedcan be incorporated into the stem of the stem loop. In anotherembodiment, a TEE can be incorporated on the 5′ end of the stem of astem loop, a miRNA seed can be incorporated into the stem of the stemloop and a miRNA binding site can be incorporated into the 3′ end of thestem or the sequence after the stem loop. The miRNA seed and the miRNAbinding site can be for the same and/or different miRNA sequences.

In one embodiment, the incorporation of a miRNA sequence and/or a TEEsequence changes the shape of the stem loop region which can increaseand/or decrease translation. (see e.g., Kedde et al., “A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility.” Nature Cell Biology. 2010, incorporated herein byreference in its entirety).

In one embodiment, the 5′-UTR of a polynucleotide of the invention cancomprise at least one miRNA sequence. The miRNA sequence can be, but isnot limited to, a 19 or 22 nucleotide sequence and/or a miRNA sequencewithout the seed.

In one embodiment the miRNA sequence in the 5′UTR can be used tostabilize a polynucleotide of the invention described herein.

In another embodiment, a miRNA sequence in the 5′UTR of a polynucleotideof the invention can be used to decrease the accessibility of the siteof translation initiation such as, but not limited to a start codon.See, e.g., Matsuda et al., PLoS One. 2010 11(5):e15057; incorporatedherein by reference in its entirety, which used antisense locked nucleicacid (LNA) oligonucleotides and exon junction complexes (EJCs) around astart codon (−4 to +37 where the A of the AUG codons is +1) in order todecrease the accessibility to the first start codon (AUG). Matsudashowed that altering the sequence around the start codon with an LNA orEJC affected the efficiency, length and structural stability of apolynucleotide. A polynucleotide of the invention can comprise a miRNAsequence, instead of the LNA or EJC sequence described by Matsuda et al,near the site of translation initiation in order to decrease theaccessibility to the site of translation initiation. The site oftranslation initiation can be prior to, after or within the miRNAsequence. As a non-limiting example, the site of translation initiationcan be located within a miRNA sequence such as a seed sequence orbinding site.

In some embodiments, a polynucleotide of the invention can include atleast one miRNA in order to dampen the antigen presentation by antigenpresenting cells. The miRNA can be the complete miRNA sequence, themiRNA seed sequence, the miRNA sequence without the seed, or acombination thereof. As a non-limiting example, a miRNA incorporatedinto a polynucleotide of the invention can be specific to thehematopoietic system. As another non-limiting example, a miRNAincorporated into a polynucleotide of the invention to dampen antigenpresentation is miR-142-3p.

In some embodiments, a polynucleotide of the invention can include atleast one miRNA in order to dampen expression of the encoded polypeptidein a tissue or cell of interest. As a non-limiting example apolynucleotide of the invention can include at least one miR-142-3pbinding site, miR-142-3p seed sequence, miR-142-3p binding site withoutthe seed, miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5pbinding site without the seed, miR-146 binding site, miR-146 seedsequence and/or miR-146 binding site without the seed sequence.

In some embodiments, a polynucleotide of the invention can comprise atleast one miRNA binding site in the 3′UTR in order to selectivelydegrade mRNA therapeutics in the immune cells to subdue unwantedimmunogenic reactions caused by therapeutic delivery. As a non-limitingexample, the miRNA binding site can make a polynucleotide of theinvention more unstable in antigen presenting cells. Non-limitingexamples of these miRNAs include mir-142-5p, mir-142-3p, mir-146a-5p,and mir-146-3p.

In one embodiment, a polynucleotide of the invention comprises at leastone miRNA sequence in a region of the polynucleotide that can interactwith a RNA binding protein.

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprising (i) a sequence-optimized nucleotide sequence(e.g., an ORF) encoding a Citrin polypeptide (e.g., the wild-typesequence, functional fragment, or variant thereof) and (ii) a miRNAbinding site (e.g., a miRNA binding site that binds to miR-142 and/or amiRNA binding site that binds to miR-126).

In some embodiments, the polynucleotide of the invention comprises auracil-modified sequence encoding a Citrin polypeptide disclosed hereinand a miRNA binding site disclosed herein, e.g., a miRNA binding sitethat binds to miR-142 and/or a miRNA binding site that binds to miR-126.In some embodiments, the polynucleotide of the invention comprises auracil-modified sequence encoding a polypeptide disclosed herein and amiRNA binding site disclosed herein, e.g., a miRNA binding site thatbinds to miR-142miR-126, miR-142, miR-144, miR-146, miR-150, miR-155,miR-16, miR-21, miR-223, miR-24, miR-27 or miR-26a. In some embodiments,the miRNA binding site binds to miR126-3p, miR-142-3p, miR-142-5p, ormiR-155. In some embodiments, the polynucleotide of the inventioncomprises a uracil-modified sequence encoding a polypeptide disclosedherein and at least two different microRNA binding sites, wherein themicroRNA is expressed in an immune cell of hematopoietic lineage or acell that expresses TLR7 and/or TLR8 and secretes pro-inflammatorycytokines and/or chemokines, and wherein the polynucleotide comprisesone or more modified nucleobases. In some embodiments, theuracil-modified sequence encoding a Citrin polypeptide comprises atleast one chemically modified nucleobase, e.g., 5-methoxyuracil. In someembodiments, at least 95% of a type of nucleobase (e.g., uricil) in auracil-modified sequence encoding a Citrin polypeptide of the inventionare modified nucleobases. In some embodiments, at least 95% of uricil ina uracil-modified sequence encoding a Citrin polypeptide is5-methoxyuridine. In some embodiments, the polynucleotide (e.g., a RNA,e.g., an mRNA) disclosed herein, e.g., comprising an miRNA binding site,is formulated with a delivery agent comprising, e.g., a compound havingthe Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; acompound having the Formula (III), (IV), (V), or (VI), e.g., any ofCompounds 233-342, e.g., Compound 236; or a compound having the Formula(VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or anycombination thereof. In some embodiments, the delivery agent comprisesCompound 18, DSPC, Cholesterol, and Compound 428, e.g., with a moleratio of about 50:10:38.5:1.5.

13. 3′ UTRs

In certain embodiments, a polynucleotide of the present invention (e.g.,a polynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide of the invention) further comprises a 3′ UTR.

3′-UTR is the section of mRNA that immediately follows the translationtermination codon and often contains regulatory regions thatpost-transcriptionally influence gene expression. Regulatory regionswithin the 3′-UTR can influence polyadenylation, translation efficiency,localization, and stability of the mRNA. In one embodiment, the 3′-UTRuseful for the invention comprises a binding site for regulatoryproteins or microRNAs. In certain embodiments, the 3′ UTR useful for thepolynucleotides of the invention comprises a 3′UTR selected from SEQ IDNOs: 48-72, 80, 81, 102-105, 108-117, 124, 125, 147-157, and anycombination thereof. In some embodiments, the 3′ UTR comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 105,147, 148, or any combination thereof. In some embodiments, the 3′ UTRcomprises a nucleic acid sequence of SEQ ID NO: 147. In someembodiments, the 3′ UTR comprises a nucleic acid sequence of SEQ ID NO:148.

In certain embodiments, the 3′ UTR sequence useful for the inventioncomprises a nucleotide sequence at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or about 100% identical to a sequence selected from the group consistingof 3′UTR sequences of SEQ ID NOs: 48-72, 80, 81, 102-105, 108-117, 124,125, 147-157, and any combination thereof.

14. Regions Having a 5′ Cap

The invention also includes a polynucleotide that comprises both a 5′Cap and a polynucleotide of the present invention (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide).

The 5′ cap structure of a natural mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns during mRNA splicing.

Endogenous mRNA molecules can be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap can then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA canoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure can target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

In some embodiments, the polynucleotides of the present invention (e.g.,a polynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) incorporate a cap moiety.

In some embodiments, polynucleotides of the present invention (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) comprise a non-hydrolyzable cap structure preventingdecapping and thus increasing mRNA half-life. Because cap structurehydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages,modified nucleotides can be used during the capping reaction. Forexample, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich,Mass.) can be used with α-thio-guanosine nucleotides according to themanufacturer's instructions to create a phosphorothioate linkage in the5′-ppp-5′ cap. Additional modified guanosine nucleotides can be usedsuch as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the polynucleotide (as mentioned above)on the 2′-hydroxyl group of the sugar ring. Multiple distinct 5′-capstructures can be used to generate the 5′-cap of a nucleic acidmolecule, such as a polynucleotide that functions as an mRNA molecule.Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e., endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs can be chemically (i.e., non-enzymatically) orenzymatically synthesized and/or linked to the polynucleotides of theinvention.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which can equivalently be designated 3′ 0-Me-m7G(5′)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped polynucleotide. The N7- and 3′-O-methylatedguanine provides the terminal moiety of the capped polynucleotide.

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

In some embodiments, the cap is a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog can be modified atdifferent phosphate positions with a boranophosphate group or aphosphoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the cap is a cap analog is aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analogknown in the art and/or described herein. Non-limiting examples of aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analoginclude a N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m^(3′-O)G(5)ppp(5′)G cap analog (See, e.g.,the various cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by referencein its entirety). In another embodiment, a cap analog of the presentinvention is a 4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotideor a region thereof, in an in vitro transcription reaction, up to 20% oftranscripts can remain uncapped. This, as well as the structuraldifferences of a cap analog from an endogenous 5′-cap structures ofnucleic acids produced by the endogenous, cellular transcriptionmachinery, can lead to reduced translational competency and reducedcellular stability.

Polynucleotides of the invention (e.g., a polynucleotide comprising anucleotide sequence encoding a Citrin polypeptide) can also be cappedpost-manufacture (whether IVT or chemical synthesis), using enzymes, inorder to generate more authentic 5′-cap structures. As used herein, thephrase “more authentic” refers to a feature that closely mirrors ormimics, either structurally or functionally, an endogenous or wild typefeature. That is, a “more authentic” feature is better representative ofan endogenous, wild-type, natural or physiological cellular functionand/or structure as compared to synthetic features or analogs, etc., ofthe prior art, or which outperforms the corresponding endogenous,wild-type, natural or physiological feature in one or more respects.Non-limiting examples of more authentic 5′cap structures of the presentinvention are those that, among other things, have enhanced binding ofcap binding proteins, increased half-life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of apolynucleotide and a guanine cap nucleotide wherein the cap guaninecontains an N7 methylation and the 5′-terminal nucleotide of the mRNAcontains a 2′-O-methyl. Such a structure is termed the Cap1 structure.This cap results in a higher translational-competency and cellularstability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′cap analog structures known inthe art. Cap structures include, but are not limited to, 7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and7mG(5′)-ppp(5′)NlmpN2mp (cap 2).

As a non-limiting example, capping chimeric polynucleotidespost-manufacture can be more efficient as nearly 100% of the chimericpolynucleotides can be capped. This is in contrast to ˜80% when a capanalog is linked to a chimeric polynucleotide in the course of an invitro transcription reaction.

According to the present invention, 5′ terminal caps can includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap can comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

15. Poly-A Tails

In some embodiments, the polynucleotides of the present disclosure(e.g., a polynucleotide comprising a nucleotide sequence encoding aCitrin polypeptide) further comprise a poly-A tail. In furtherembodiments, terminal groups on the poly-A tail can be incorporated forstabilization. In other embodiments, a poly-A tail comprises des-3′hydroxyl tails.

During RNA processing, a long chain of adenine nucleotides (poly-A tail)can be added to a polynucleotide such as an mRNA molecule in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript can be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between, for example,approximately 80 to approximately 250 residues long, includingapproximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240 or 250 residues long.

PolyA tails can also be added after the construct is exported from thenucleus.

According to the present invention, terminal groups on the poly A tailcan be incorporated for stabilization. Polynucleotides of the presentinvention can include des-3′ hydroxyl tails. They can also includestructural moieties or 2′-Omethyl modifications as taught by Junjie Li,et al. (Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005, the contentsof which are incorporated herein by reference in its entirety).

The polynucleotides of the present invention can be designed to encodetranscripts with alternative polyA tail structures including histonemRNA. According to Norbury, “Terminal uridylation has also been detectedon human replication-dependent histone mRNAs. The turnover of thesemRNAs is thought to be important for the prevention of potentially toxichistone accumulation following the completion or inhibition ofchromosomal DNA replication. These mRNAs are distinguished by their lackof a 3′ poly(A) tail, the function of which is instead assumed by astable stem-loop structure and its cognate stem-loop binding protein(SLBP); the latter carries out the same functions as those of PABP onpolyadenylated mRNAs” (Norbury, “Cytoplasmic RNA: a case of the tailwagging the dog,” Nature Reviews Molecular Cell Biology; AOP, publishedonline 29 Aug. 2013; doi:10.1038/nrm3645) the contents of which areincorporated herein by reference in its entirety.

Unique poly-A tail lengths provide certain advantages to thepolynucleotides of the present invention. Generally, the length of apoly-A tail, when present, is greater than 30 nucleotides in length. Inanother embodiment, the poly-A tail is greater than 35 nucleotides inlength (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700,1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).

In some embodiments, the polynucleotide or region thereof includes fromabout 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000,from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100,from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750,from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500,and from 2,500 to 3,000).

In some embodiments, the poly-A tail is designed relative to the lengthof the overall polynucleotide or the length of a particular region ofthe polynucleotide. This design can be based on the length of a codingregion, the length of a particular feature or region or based on thelength of the ultimate product expressed from the polynucleotides.

In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotide or featurethereof. The poly-A tail can also be designed as a fraction of thepolynucleotides to which it belongs. In this context, the poly-A tailcan be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the totallength of the construct, a construct region or the total length of theconstruct minus the poly-A tail. Further, engineered binding sites andconjugation of polynucleotides for Poly-A binding protein can enhanceexpression.

Additionally, multiple distinct polynucleotides can be linked togethervia the PABP (Poly-A binding protein) through the 3′-end using modifiednucleotides at the 3′-terminus of the poly-A tail. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day7 post-transfection.

In some embodiments, the polynucleotides of the present invention aredesigned to include a polyA-G Quartet region. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultantpolynucleotide is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein production froman mRNA equivalent to at least 75% of that seen using a poly-A tail of120 nucleotides alone.

16. Start Codon Region

The invention also includes a polynucleotide that comprises both a startcodon region and the polynucleotide described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide). In some embodiments, the polynucleotides of the presentinvention can have regions that are analogous to or function like astart codon region.

In some embodiments, the translation of a polynucleotide can initiate ona codon that is not the start codon AUG. Translation of thepolynucleotide can initiate on an alternative start codon such as, butnot limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU,TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 andMatsuda and Mauro PLoS ONE, 2010 5:11; the contents of each of which areherein incorporated by reference in its entirety).

As a non-limiting example, the translation of a polynucleotide begins onthe alternative start codon ACG. As another non-limiting example,polynucleotide translation begins on the alternative start codon CTG orCUG. As yet another non-limiting example, the translation of apolynucleotide begins on the alternative start codon GTG or GUG.

Nucleotides flanking a codon that initiates translation such as, but notlimited to, a start codon or an alternative start codon, are known toaffect the translation efficiency, the length and/or the structure ofthe polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 2010 5:11;the contents of which are herein incorporated by reference in itsentirety). Masking any of the nucleotides flanking a codon thatinitiates translation can be used to alter the position of translationinitiation, translation efficiency, length and/or structure of apolynucleotide.

In some embodiments, a masking agent can be used near the start codon oralternative start codon in order to mask or hide the codon to reduce theprobability of translation initiation at the masked start codon oralternative start codon. Non-limiting examples of masking agents includeantisense locked nucleic acids (LNA) polynucleotides and exon junctioncomplexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agentsLNA polynucleotides and EJCs (PLoS ONE, 2010 5:11); the contents ofwhich are herein incorporated by reference in its entirety).

In another embodiment, a masking agent can be used to mask a start codonof a polynucleotide in order to increase the likelihood that translationwill initiate on an alternative start codon. In some embodiments, amasking agent can be used to mask a first start codon or alternativestart codon in order to increase the chance that translation willinitiate on a start codon or alternative start codon downstream to themasked start codon or alternative start codon.

In some embodiments, a start codon or alternative start codon can belocated within a perfect complement for a miRNA binding site. Theperfect complement of a miRNA binding site can help control thetranslation, length and/or structure of the polynucleotide similar to amasking agent. As a non-limiting example, the start codon or alternativestart codon can be located in the middle of a perfect complement for amiRNA binding site. The start codon or alternative start codon can belocated after the first nucleotide, second nucleotide, third nucleotide,fourth nucleotide, fifth nucleotide, sixth nucleotide, seventhnucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide,eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide,fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide,seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide,twentieth nucleotide or twenty-first nucleotide.

In another embodiment, the start codon of a polynucleotide can beremoved from the polynucleotide sequence in order to have thetranslation of the polynucleotide begin on a codon that is not the startcodon. Translation of the polynucleotide can begin on the codonfollowing the removed start codon or on a downstream start codon or analternative start codon. In a non-limiting example, the start codon ATGor AUG is removed as the first 3 nucleotides of the polynucleotidesequence in order to have translation initiate on a downstream startcodon or alternative start codon. The polynucleotide sequence where thestart codon was removed can further comprise at least one masking agentfor the downstream start codon and/or alternative start codons in orderto control or attempt to control the initiation of translation, thelength of the polynucleotide and/or the structure of the polynucleotide.

17. Stop Codon Region

The invention also includes a polynucleotide that comprises both a stopcodon region and the polynucleotide described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide). In some embodiments, the polynucleotides of the presentinvention can include at least two stop codons before the 3′untranslated region (UTR). The stop codon can be selected from TGA, TAAand TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA.In some embodiments, the polynucleotides of the present inventioninclude the stop codon TGA in the case or DNA, or the stop codon UGA inthe case of RNA, and one additional stop codon. In a further embodimentthe addition stop codon can be TAA or UAA. In another embodiment, thepolynucleotides of the present invention include three consecutive stopcodons, four stop codons, or more.

18. Insertions and Substitutions

The invention also includes a polynucleotide of the present disclosurethat further comprises insertions and/or substitutions.

In some embodiments, the 5′UTR of the polynucleotide can be replaced bythe insertion of at least one region and/or string of nucleosides of thesame base. The region and/or string of nucleotides can include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides can be natural and/orunnatural. As a non-limiting example, the group of nucleotides caninclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In some embodiments, the 5′UTR of the polynucleotide can be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR can be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR can be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In some embodiments, the polynucleotide can include at least onesubstitution and/or insertion downstream of the transcription start sitethat can be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion can occur downstream of thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site can affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleoside can cause a silent mutation of the sequence or cancause a mutation in the amino acid sequence.

In some embodiments, the polynucleotide can include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12 or at least 13 guanine bases downstream of the transcription startsite.

In some embodiments, the polynucleotide can include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA, the guanine bases can be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basescan be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases can be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In some embodiments, the polynucleotide can include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Thepolynucleotide can include, but is not limited to, at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7 or atleast 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases can be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted can be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases.

As a non-limiting example, the guanine base upstream of the codingregion in the polynucleotide can be substituted with adenine, cytosine,thymine, or any of the nucleotides described herein. In anothernon-limiting example the substitution of guanine bases in thepolynucleotide can be designed so as to leave one guanine base in theregion downstream of the transcription start site and before the startcodon (see Esvelt et al. Nature (2011) 472(7344):499-503; the contentsof which is herein incorporated by reference in its entirety). As anon-limiting example, at least 5 nucleotides can be inserted at 1location downstream of the transcription start site but upstream of thestart codon and the at least 5 nucleotides can be the same base type.

19. Polynucleotide Comprising an mRNA Encoding a Citrin Polypeptide

In certain embodiments, a polynucleotide of the present disclosure, forexample a polynucleotide comprising an mRNA nucleotide sequence encodinga Citrin polypeptide, comprises from 5′ to 3′ end:

-   -   (i) a 5′ cap provided above;    -   (ii) a 5′ UTR, such as the sequences provided above;    -   (iii) an open reading frame encoding a Citrin polypeptide, e.g.,        a sequence optimized nucleic acid sequence encoding Citrin        disclosed herein;    -   (iv) at least one stop codon;    -   (v) a 3′ UTR, such as the sequences provided above; and    -   (vi) a poly-A tail provided above.

In some embodiments, the polynucleotide further comprises a miRNAbinding site, e.g., a miRNA binding site that binds to miRNA-142. Insome embodiments, the 5′UTR comprises the miRNA binding site.

In some embodiments, a polynucleotide of the present disclosurecomprises a nucleotide sequence encoding a polypeptide sequence at least70%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to the protein sequence of a wild type Citrin(e.g., isoform 1 or 2).

In some embodiments, a polynucleotide of the present disclosure, forexample a polynucleotide comprising an mRNA nucleotide sequence encodinga Citrin polypeptide, comprises (1) a 5′ cap provided above, forexample, CAP1, (2) a nucleotide sequence selected form the groupconsisting of SEQ ID NO: 130, 133, 136, and 139, and (3) a poly-A tailprovided above, for example, a poly A tail of about 100 residues,wherein

-   -   SEQ ID NO: 130 comprises from 5′ to 3′ end: 5′ UTR of SEQ ID        NO:30, CTNL2 polypeptide ORF of SEQ ID NO: 129, and 3′UTR of SEQ        ID NO: 105;    -   SEQ ID NO: 133 comprises from 5′ to 3′ end: 5′ UTR of SEQ ID        NO:30, CTNL2 polypeptide ORF of SEQ ID NO: 132, and 3′UTR of SEQ        ID NO: 105;    -   SEQ ID NO: 136 comprises from 5′ to 3′ end: 5′ UTR of SEQ ID        NO:30, CTNL2 polypeptide ORF of SEQ ID NO: 135, and 3′UTR of SEQ        ID NO: 105;    -   SEQ ID NO: 139 comprises from 5′ to 3′ end: 5′ UTR of SEQ ID        NO:30, CTNL2 polypeptide ORF of SEQ ID NO: 138, and 3′UTR of SEQ        ID NO: 105.

TABLE 5 mRNA Constructs SEQ ID ConstructSequence (5′UTR = bold underline; 3′UTR comprising  NO. no.a stop codon = bold italics) 130 #1

AUG GCCGCCGCCAAGGUCGCCCUCACCAAGAGGGCGGACCCCGCGGAGCUCAGGACCAUCUUUCUCAAGUACGCCUCCAUCGAGAAGAACGGGGAGUUCUUCAUGAGCCCGAACGAUUUCGUCACCAGGUACCUCAACAUCUUCGGCGAGAGCCAGCCCAACCCGAAGACCGUAGAGCUCCUCAGCGGCGUCGUCGACCAGACGAAGGACGGACUCAUUAGCUUCCAGGAGUUCGUCGCCUUCGAGAGCGUCCUCUGCGCGCCCGACGCCCUCUUCAUGGUCGCGUUCCAGCUGUUCGAUAAGGCCGGCAAGGGCGAGGUGACCUUUGAAGAUGUGAAGCAGGUGUUCGGUCAGACCACCAUCCACCAGCACAUCCCUUUUAACUGGGACUCAGAAUUCGUCCAGCUGCACUUCGGCAAGGAAAGGAAGCGCCAUCUGACCUACGCCGAGUUCACACAGUUUCUCCUGGAGAUCCAGCUGGAACACGCCAAACAGGCGUUCGUUCAGCGGGACAACGCCAGGACCGGCCGGGUCACCGCGAUCGACUUCCGUGAUAUCAUGGUGACCAUCAGGCCUCACGUCCUGACGCCCUUCGUGGAAGAGUGCCUGGUGGCCGCCGCGGGUGGGACCACGUCCCACCAGGUGAGCUUCUCCUAUUUUAACGGGUUCAACAGCCUCCUCAACAACAUGGAGCUGAUCCGGAAGAUCUAUAGCACCCUGGCCGGGACGCGCAAGGACGUCGAGGUGACCAAGGAAGAAUUCGUGCUGGCCGCCCAGAAGUUUGGCCAGGUGACGCCCAUGGAGGUGGACAUCCUGUUCCAACUGGCGGACCUGUACGAACCCAGGGGCCGGAUGACCCUGGCCGACAUCGAGCGCAUUGCCCCGCUCGAGGAGGGCACCCUGCCCUUCAACCUGGCGGAGGCCCAGCGCCAGAAGGCCAGCGGGGACAGCGCCAGGCCCGUCCUGCUGCAGGUGGCCGAGUCCGCGUACAGGUUCGGUCUGGGGAGCGUCGCGGGAGCCGUGGGUGCCACCGCCGUGUAUCCGAUCGACCUCGUGAAGACCCGUAUGCAGAAUCAGAGGUCCACCGGCAGCUUCGUGGGCGAGCUCAUGUACAAGAACUCCUUCGACUGUUUCAAGAAGGUUCUCCGGUACGAGGGGUUCUUCGGCCUCUACCGGGGACUGCUACCCCAGCUGCUCGGCGUGGCGCCCGAGAAGGCCAUCAAGCUCACGGUCAACGACUUUGUGCGGGACAAGUUCAUGCACAAGGACGGCAGCGUCCCACUGGCCGCCGAGAUUCUGGCAGGCGGCUGCGCCGGAGGCAGCCAGGUCAUCUUUACCAACCCACUGGAGAUCGUGAAGAUCAGGCUGCAAGUGGCCGGGGAGAUUACCACCGGACCCCGUGUGAGCGCCCUGAGCGUGGUGAGGGACCUUGGCUUCUUUGGCAUAUACAAGGGAGCCAAGGCCUGCUUUCUGAGGGACAUCCCGUUUUCCGCCAUAUACUUUCCCUGCUACGCCCACGUGAAAGCGAGCUUCGCCAACGAGGACGGCCAGGUCAGUCCCGGCUCCCUACUGCUGGCCGGAGCCAUCGCCGGGAUGCCCGCCGCCAGCCUGGUGACGCCCGCGGACGUUAUCAAGACCAGGCUGCAGGUGGCGGCCAGGGCGGGCCAGACCACCUACAGCGGCGUGAUCGACUGCUUCCGCAAGAUCCUGCGGGAGGAGGGGCCCAAGGCCCUGUGGAAAGGCGCCGGGGCAAGGGUGUUCAGGAGCAGCCCACAGUUCGGGGUGACCCUGCUUACCUACGAACUGCUGCAGCGGUGGUUCUAUAUCGACUUCGGAGGAGUCAAGCCCAUGGGCUCCGAGCCUGUGCCCAAGAGCAGGAUCAAUCUGCCCGCGCCCAACCCAGACCACGUGGGAGGGUACAAACUGGCCGUGGCCACCUUCGCCGGAAUCGAGAACAAGUUCGGCCUGUAUCUGCCCCUGUUCAAACCGUCCGUCUCCACCAGCAAGGCCAUCGGCGGAGGCCCC

133 #2

AUG GCCGCCGCCAAGGUCGCCCUCACCAAGAGGGCGGACCCCGCGGAGCUCAGGACCAUCUUUCUCAAGUACGCCUCCAUCGAGAAGAACGGGGAGUUCUUCAUGAGCCCGAACGAUUUCGUCACCAGGUACCUCAACAUCUUCGGCGAGAGCCAGCCCAACCCGAAGACCGUAGAGCUCCUCAGCGGCGUCGUCGACCAGACGAAGGACGGACUCAUUAGCUUCCAGGAGUUCGUCGCCUUCGAGAGCGUCCUCUGCGCCCCCGACGCCCUCUUCAUGGUCGCGUUCCAGCUGUUCGAUAAGGCCGGCAAGGGCGAGGUGACCUUUGAAGAUGUGAAGCAGGUGUUCGGUCAGACCACCAUCCACCAGCACAUCCCUUUUAACUGGGACUCAGAAUUCGUCCAGCUGCACUUCGGCAAGGAAAGGAAGCGCCAUCUGACCUACGCCGAGUUCACACAGUUUCUCCUGGAGAUCCAGCUGGAACACGCCAAACAGGCGUUCGUUCAGCGGGACAACGCCAGGACCGGCCGGGUCACCGCGAUCGACUUCCGUGAUAUCAUGGUGACCAUCAGGCCUCACGUCCUGACCCCCUUCGUGGAAGAGUGCCUGGUGGCCGCCGCGGGUGGGACCACGUCCCACCAGGUGAGCUUCUCCUAUUUUAACGGGUUCAACAGCCUCCUCAACAACAUGGAGCUGAUCCGGAAGAUCUAUAGCACCCUGGCCGGGACGCGCAAGGACGUCGAGGUGACCAAGGAAGAAUUCGUGCUGGCCGCCCAGAAGUUUGGCCAGGUGACCCCCAUGGAGGUGGACAUCCUGUUCCAACUGGCGGACCUGUACGAACCCAGGGGCCGGAUGACCCUGGCCGACAUCGAGCGCAUUGCCCCGCUCGAGGAGGGCACCCUGCCCUUCAACCUGGCGGAGGCCCAGCGCCAGAAGGCCAGCGGGGACAGCGCCAGGCCCGUCCUGCUGCAGGUGGCCGAGUCCGCGUACAGGUUCGGUCUGGGGAGCGUCGCGGGAGCCGUGGGUGCCACCGCCGUGUAUCCGAUCGACCUCGUGAAGACCCGUAUGCAGAAUCAGAGGUCCACCGGCAGCUUCGUGGGCGAGCUCAUGUACAAGAACUCCUUCGACUGUUUCAAGAAGGUUCUCCGGUACGAGGGGUUCUUCGGCCUCUACCGGGGGCUGCUACCCCAGCUGCUCGGCGUGGCGCCCGAGAAGGCCAUCAAGCUCACGGUCAACGACUUUGUGCGGGACAAGUUCAUGCACAAGGACGGCAGCGUCCCCCUGGCCGCCGAGAUUCUGGCAGGCGGCUGCGCCGGAGGCAGCCAGGUCAUCUUUACCAACCCCCUGGAGAUCGUGAAGAUCAGGCUGCAAGUGGCCGGGGAGAUUACCACCGGACCCCGUGUGAGCGCCCUGAGCGUGGUGAGGGACCUUGGCUUCUUUGGCAUAUACAAGGGAGCCAAGGCCUGCUUUCUGAGGGACAUCCCGUUUUCCGCCAUAUACUUUCCCUGCUACGCCCACGUGAAAGCGAGCUUCGCCAACGAGGACGGCCAGGUCAGUCCCGGCUCCCUACUGCUGGCCGGAGCCAUCGCCGGGAUGCCCGCCGCCAGCCUGGUGACCCCCGCGGACGUUAUCAAGACCAGGCUGCAGGUGGCGGCCAGGGCGGGCCAGACCACCUACAGCGGCGUGAUCGACUGCUUCCGCAAGAUCCUGCGGGAGGAGGGGCCCAAGGCCCUGUGGAAAGGCGCCGGGGCAAGGGUGUUCAGGAGCAGCCCCCAGUUCGGGGUGACCCUGCUUACCUACGAACUGCUGCAGCGGUGGUUCUAUAUCGACUUCGGGGGAGUCAAGCCCAUGGGCUCCGAGCCUGUGCCCAAGAGCAGGAUCAAUCUGCCCGCCCCCAACCCAGACCACGUGGGAGGGUACAAACUGGCCGUGGCCACCUUCGCCGGAAUCGAGAACAAGUUCGGCCUGUAUCUGCCCCUGUUCAAACCGUCCGUCUCCACCAGCAAGGCCAUCGGCGGGGGCCCC

136 #3

AUG GCCGCCGCCAAGGUCGCACUCACCAAACGGGCCGACCCCGCCGAGCUCAGGACCAUCUUCCUCAAGUACGCCAGCAUCGAGAAGAACGGGGAGUUCUUCAUGAGCCCCAACGACUUCGUAACCAGGUACCUCAAUAUCUUCGGCGAAAGCCAGCCCAACCCGAAGACCGUCGAGCUCCUCUCCGGGGUCGUCGAUCAGACCAAGGACGGGCUCAUCUCCUUCCAGGAGUUCGUGGCCUUCGAGUCCGUCCUCUGCGCCCCGGACGCCCUCUUCAUGGUCGCCUUCCAACUCUUCGAUAAGGCCGGGAAAGGCGAGGUGACCUUCGAAGAUGUGAAGCAGGUUUUCGGCCAGACCACGAUCCACCAGCAUAUCCCCUUUAACUGGGACAGCGAGUUCGUGCAGCUGCACUUCGGCAAAGAGAGGAAGAGGCACCUCACCUAUGCCGAAUUCACACAAUUCCUGCUCGAGAUUCAGCUGGAGCAUGCCAAACAAGCCUUUGUCCAGCGGGACAACGCCAGGACCGGCAGGGUCACCGCGAUCGACUUUCGCGAUAUCAUGGUGACCAUCAGGCCCCACGUGCUCACCCCCUUUGUGGAGGAGUGCCUGGUCGCCGCCGCCGGCGGGACGACCAGCCACCAGGUGUCCUUCUCAUACUUCAACGGGUUCAACAGCCUGCUGAACAACAUGGAGCUGAUCCGCAAGAUCUACUCCACCCUGGCCGGCACCAGGAAGGACGUGGAAGUGACCAAGGAGGAGUUCGUUCUCGCCGCCCAGAAAUUCGGACAGGUGACCCCGAUGGAGGUGGACAUCCUCUUCCAGCUGGCCGAUCUCUACGAGCCCCGGGGCAGGAUGACGCUCGCCGACAUCGAGCGAAUCGCCCCCCUGGAGGAGGGUACCCUUCCCUUCAACCUGGCCGAGGCCCAGCGACAGAAAGCCAGCGGCGACAGUGCCAGGCCCGUGCUCCUGCAGGUGGCCGAAUCCGCCUACAGAUUUGGCCUGGGCAGCGUCGCCGGCGCAGUGGGCGCCACCGCCGUGUACCCGAUCGACCUGGUCAAGACCAGGAUGCAGAACCAGAGAAGCACCGGCUCCUUUGUGGGCGAGCUUAUGUACAAGAACAGCUUCGACUGCUUCAAGAAGGUCCUGCGUUACGAGGGCUUCUUCGGGCUGUACCGGGGCCUGCUGCCGCAGCUGCUAGGCGUCGCGCCCGAGAAGGCCAUUAAGCUGACCGUGAACGACUUCGUACGGGACAAAUUCAUGCACAAGGACGGCAGUGUGCCCCUGGCCGCAGAGAUCCUGGCCGGGGGCUGUGCCGGCGGGUCCCAGGUGAUCUUUACGAACCCCCUGGAGAUAGUGAAGAUCCGGCUGCAGGUGGCCGGGGAGAUCACCACCGGCCCCCGGGUGUCCGCCCUGAGCGUCGUGAGGGAUCUGGGGUUCUUUGGCAUCUAUAAGGGUGCCAAGGCCUGCUUCCUGCGUGACAUUCCCUUUAGCGCCAUCUACUUCCCCUGCUACGCCCAUGUCAAGGCCAGCUUCGCCAACGAAGACGGACAGGUGAGCCCCGGGUCCCUGCUGCUGGCCGGCGCCAUCGCAGGGAUGCCGGCCGCCUCACUGGUGACCCCGGCGGACGUGAUAAAGACGAGGCUGCAGGUGGCCGCCCGCGCAGGCCAGACCACGUACUCGGGUGUGAUAGACUGCUUCCGGAAGAUCCUGAGGGAAGAAGGCCCCAAGGCCCUGUGGAAGGGGGCCGGCGCCAGGGUGUUCCGCAGCAGCCCCCAGUUCGGGGUCACCCUGCUCACGUACGAGCUCCUGCAGAGGUGGUUCUACAUCGACUUUGGCGGGGUCAAGCCCAUGGGCAGUGAGCCGGUGCCCAAGAGCCGGAUCAAUCUGCCCGCCCCCAACCCUGACCACGUCGGCGGGUACAAACUGGCGGUGGCCACCUUCGCCGGCAUCGAGAACAAGUUUGGCCUGUACCUGCCGCUGUUUAAGCCGUCCGUGAGCACCUCCAAGGCCAUCGGGGGCGGCCCA

139 #4

AUG GCCGCCGCUAAGGUCGCCCUCACGAAGCGGGCCGAUCCCGCCGAGCUCCGGACCAUCUUUCUCAAAUACGCCAGCAUCGAGAAGAACGGCGAGUUCUUCAUGUCCCCCAACGACUUCGUCACCAGGUACCUAAACAUCUUCGGCGAGAGCCAGCCGAACCCCAAGACCGUCGAAUUGCUAUCGGGCGUCGUCGACCAGACCAAGGACGGGCUCAUCAGCUUCCAGGAGUUCGUCGCCUUCGAGUCCGUCCUCUGCGCCCCCGACGCCCUCUUCAUGGUAGCAUUCCAGUUGUUCGACAAGGCCGGAAAGGGGGAGGUGACCUUCGAGGACGUGAAGCAGGUGUUUGGUCAGACCACCAUCCAUCAGCACAUCCCGUUUAACUGGGACUCGGAGUUCGUGCAGCUCCACUUCGGCAAGGAACGAAAGCGGCACCUCACCUACGCCGAGUUCACCCAGUUCCUGCUGGAGAUCCAGCUGGAACACGCCAAGCAGGCCUUCGUGCAGCGAGACAAUGCGAGGACCGGGCGCGUGACUGCCAUCGAUUUCAGGGACAUCAUGGUGACCAUCCGGCCCCACGUGCUGACUCCCUUCGUGGAGGAGUGCCUGGUCGCAGCCGCCGGGGGUACCACCUCCCACCAGGUGAGCUUCAGCUACUUCAACGGCUUCAACAGCCUGCUCAACAACAUGGAACUGAUCCGCAAGAUCUAUUCCACACUCGCCGGCACCAGGAAGGAUGUGGAGGUGACCAAGGAGGAGUUUGUGCUGGCGGCCCAGAAGUUUGGGCAGGUCACCCCCAUGGAGGUGGACAUCCUCUUCCAGCUGGCCGACCUGUACGAGCCCAGGGGCAGGAUGACCCUGGCCGACAUCGAACGAAUCGCCCCCCUGGAGGAGGGCACACUGCCCUUCAACCUGGCCGAGGCCCAGAGGCAGAAGGCCUCCGGCGACAGCGCCCGACCCGUGCUUCUGCAAGUGGCCGAGAGCGCCUACCGCUUUGGCCUGGGAUCCGUGGCCGGCGCCGUCGGUGCCACCGCCGUGUACCCAAUCGACCUAGUGAAGACACGCAUGCAGAACCAGCGCAGCACCGGCUCCUUUGUCGGGGAGCUGAUGUAUAAGAACAGCUUCGACUGCUUCAAGAAGGUGCUGCGCUACGAGGGCUUCUUCGGCCUGUACAGGGGGCUGCUGCCCCAACUGCUGGGCGUGGCCCCCGAGAAGGCCAUCAAGCUGACCGUUAACGACUUCGUGCGAGACAAGUUCAUGCACAAGGACGGCAGCGUGCCCUUGGCCGCAGAGAUACUGGCCGGGGGCUGCGCCGGCGGGUCGCAGGUGAUCUUCACCAACCCCCUCGAGAUCGUAAAGAUCAGGCUCCAAGUGGCCGGGGAGAUCACCACCGGCCCCAGGGUGUCCGCCCUGUCGGUGGUCAGGGACCUGGGGUUCUUCGGGAUAUACAAGGGCGCGAAGGCCUGCUUCCUGAGGGACAUCCCCUUCAGCGCCAUCUACUUUCCCUGCUACGCCCACGUCAAGGCCAGCUUCGCCAACGAGGAUGGCCAGGUGUCCCCGGGCUCCCUGCUCCUGGCCGGUGCUAUCGCGGGAAUGCCCGCCGCGAGCCUGGUGACCCCCGCCGAUGUGAUCAAGACCAGGCUGCAAGUCGCGGCGAGGGCCGGUCAGACCACCUACUCCGGGGUCAUCGAUUGCUUUAGGAAGAUCCUCCGGGAGGAGGGGCCCAAGGCACUGUGGAAGGGGGCCGGGGCCAGGGUGUUCCGUAGCUCCCCCCAGUUUGGCGUGACCCUGCUGACCUACGAACUGCUGCAGAGGUGGUUCUACAUCGACUUCGGGGGCGUGAAGCCCAUGGGCUCCGAGCCGGUGCCUAAGAGCCGGAUCAAUCUGCCCGCCCCCAACCCCGACCACGUGGGGGGCUACAAGCUGGCCGUGGCUACCUUCGCCGGUAUCGAGAACAAGUUCGGCCUGUACCUGCCCCUGUUCAAGCCCUCCGUGAGCACGAGCAAGGCCAUCGGGGGCGGCCCCU

20. Methods of Making Polynucleotides

The present disclosure also provides methods for making a polynucleotideof the invention (e.g., a polynucleotide comprising a nucleotidesequence encoding a Citrin polypeptide) or a complement thereof.

In some aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosedherein, and encoding a Citrin polypeptide, can be constructed using invitro transcription. In other aspects, a polynucleotide (e.g., a RNA,e.g., an mRNA) disclosed herein, and encoding a Citrin polypeptide, canbe constructed by chemical synthesis using an oligonucleotidesynthesizer.

In other aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA)disclosed herein, and encoding a Citrin polypeptide is made by using ahost cell. In certain aspects, a polynucleotide (e.g., a RNA, e.g., anmRNA) disclosed herein, and encoding a Citrin polypeptide is made by oneor more combination of the IVT, chemical synthesis, host cellexpression, or any other methods known in the art.

Naturally occurring nucleosides, non-naturally occurring nucleosides, orcombinations thereof, can totally or partially naturally replaceoccurring nucleosides present in the candidate nucleotide sequence andcan be incorporated into a sequence-optimized nucleotide sequence (e.g.,a RNA, e.g., an mRNA) encoding a Citrin polypeptide. The resultantpolynucleotides, e.g., mRNAs, can then be examined for their ability toproduce protein and/or produce a therapeutic outcome.

a. In Vitro Transcription/Enzymatic Synthesis

The polynucleotides of the present invention disclosed herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) can be transcribed using an in vitro transcription (IVT)system. The system typically comprises a transcription buffer,nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.The NTPs can be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasecan be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate polynucleotides disclosed herein. SeeU.S. Publ. No. US20130259923, which is herein incorporated by referencein its entirety.

Any number of RNA polymerases or variants can be used in the synthesisof the polynucleotides of the present invention. RNA polymerases can bemodified by inserting or deleting amino acids of the RNA polymerasesequence. As a non-limiting example, the RNA polymerase can be modifiedto exhibit an increased ability to incorporate a 2′-modified nucleotidetriphosphate compared to an unmodified RNA polymerase (see InternationalPublication WO2008078180 and U.S. Pat. No. 8,101,385; hereinincorporated by reference in their entireties).

Variants can be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants can be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature 472:499-503 (2011);herein incorporated by reference in its entirety) where clones of T7 RNApolymerase can encode at least one mutation such as, but not limited to,lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V,V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H,F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M2671,G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R,M401T, N410S, K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K,K577E, K577M, N601S, S684Y, L6991, K713E, N748D, Q754R, E775K, A827V,D851N or L864F. As another non-limiting example, T7 RNA polymerasevariants can encode at least mutation as described in U.S. Pub. Nos.20100120024 and 20070117112; herein incorporated by reference in theirentireties. Variants of RNA polymerase can also include, but are notlimited to, substitutional variants, conservative amino acidsubstitution, insertional variants, and/or deletional variants.

In one aspect, the polynucleotide can be designed to be recognized bythe wild type or variant RNA polymerases. In doing so, thepolynucleotide can be modified to contain sites or regions of sequencechanges from the wild type or parent chimeric polynucleotide.

Polynucleotide or nucleic acid synthesis reactions can be carried out byenzymatic methods utilizing polymerases. Polymerases catalyze thecreation of phosphodiester bonds between nucleotides in a polynucleotideor nucleic acid chain. Currently known DNA polymerases can be dividedinto different families based on amino acid sequence comparison andcrystal structure analysis. DNA polymerase I (pol I) or A polymerasefamily, including the Klenow fragments of E. coli, Bacillus DNApolymerase I, Therms aquaticus (Taq) DNA polymerases, and the T7 RNA andDNA polymerases, is among the best studied of these families. Anotherlarge family is DNA polymerase α (pol α) or B polymerase family,including all eukaryotic replicating DNA polymerases and polymerasesfrom phages T4 and RB69. Although they employ similar catalyticmechanism, these families of polymerases differ in substratespecificity, substrate analog-incorporating efficiency, degree and ratefor primer extension, mode of DNA synthesis, exonuclease activity, andsensitivity against inhibitors.

DNA polymerases are also selected based on the optimum reactionconditions they require, such as reaction temperature, pH, and templateand primer concentrations. Sometimes a combination of more than one DNApolymerases is employed to achieve the desired DNA fragment size andsynthesis efficiency. For example, Cheng et al. increase pH, addglycerol and dimethyl sulfoxide, decrease denaturation times, increaseextension times, and utilize a secondary thermostable DNA polymerasethat possesses a 3′ to 5′ exonuclease activity to effectively amplifylong targets from cloned inserts and human genomic DNA. (Cheng et al.,PNAS 91:5695-5699 (1994), the contents of which are incorporated hereinby reference in their entirety). RNA polymerases from bacteriophage T3,T7, and SP6 have been widely used to prepare RNAs for biochemical andbiophysical studies. RNA polymerases, capping enzymes, and poly-Apolymerases are disclosed in the co-pending International PublicationNo. WO2014028429, the contents of which are incorporated herein byreference in their entirety.

In one aspect, the RNA polymerase which can be used in the synthesis ofthe polynucleotides of the present invention is a Syn5 RNA polymerase.(see Zhu et al. Nucleic Acids Research 2013, doi:10.1093/nar/gkt1193,which is herein incorporated by reference in its entirety). The Syn5 RNApolymerase was recently characterized from marine cyanophage Syn5 by Zhuet al. where they also identified the promoter sequence (see Zhu et al.Nucleic Acids Research 2013, the contents of which is hereinincorporated by reference in its entirety). Zhu et al. found that Syn5RNA polymerase catalyzed RNA synthesis over a wider range oftemperatures and salinity as compared to T7 RNA polymerase.Additionally, the requirement for the initiating nucleotide at thepromoter was found to be less stringent for Syn5 RNA polymerase ascompared to the T7 RNA polymerase making Syn5 RNA polymerase promisingfor RNA synthesis.

In one aspect, a Syn5 RNA polymerase can be used in the synthesis of thepolynucleotides described herein. As a non-limiting example, a Syn5 RNApolymerase can be used in the synthesis of the polynucleotide requiringa precise 3′-terminus.

In one aspect, a Syn5 promoter can be used in the synthesis of thepolynucleotides. As a non-limiting example, the Syn5 promoter can be5′-ATTGGGCACCCGTAAGGG-3′ (SEQ ID NO: 78) as described by Zhu et al.(Nucleic Acids Research 2013).

In one aspect, a Syn5 RNA polymerase can be used in the synthesis ofpolynucleotides comprising at least one chemical modification describedherein and/or known in the art (see e.g., the incorporation ofpseudo-UTP and 5Me-CTP described in Zhu et al. Nucleic Acids Research2013).

In one aspect, the polynucleotides described herein can be synthesizedusing a Syn5 RNA polymerase which has been purified using modified andimproved purification procedure described by Zhu et al. (Nucleic AcidsResearch 2013).

Various tools in genetic engineering are based on the enzymaticamplification of a target gene which acts as a template. For the studyof sequences of individual genes or specific regions of interest andother research needs, it is necessary to generate multiple copies of atarget gene from a small sample of polynucleotides or nucleic acids.Such methods can be applied in the manufacture of the polynucleotides ofthe invention. For example, polymerase chain reaction (PCR), stranddisplacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), also called transcription mediated amplification(TMA), and/or rolling-circle amplification (RCA) can be utilized in themanufacture of one or more regions of the polynucleotides of the presentinvention.

b. Chemical Synthesis

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest, such as apolynucleotide of the invention (e.g., a polynucleotide comprising anucleotide sequence encoding a Citrin polypeptide). For example, asingle DNA or RNA oligomer containing a codon-optimized nucleotidesequence coding for the particular isolated polypeptide can besynthesized. In other aspects, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.In some aspects, the individual oligonucleotides typically contain 5′ or3′ overhangs for complementary assembly.

A polynucleotide disclosed herein (e.g., a RNA, e.g., an mRNA) can bechemically synthesized using chemical synthesis methods and potentialnucleobase substitutions known in the art. See, for example,International Publication Nos. WO2014093924, WO2013052523; WO2013039857,WO2012135805, WO2013151671; U.S. Publ. No. US20130115272; or U.S. Pat.No. 8,999,380 or 8,710,200, all of which are herein incorporated byreference in their entireties.

c. Purification of Polynucleotides Encoding Citrin

Purification of the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) can include, but is not limited to, polynucleotideclean-up, quality assurance and quality control. Clean-up can beperformed by methods known in the arts such as, but not limited to,AGENCOURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-Tbeads, LNA™ oligo-T capture probes (EXIQON® Inc., Vedbaek, Denmark) orHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

The term “purified” when used in relation to a polynucleotide such as a“purified polynucleotide” refers to one that is separated from at leastone contaminant. As used herein, a “contaminant” is any substance thatmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

In some embodiments, purification of a polynucleotide of the invention(e.g., a polynucleotide comprising a nucleotide sequence encoding aCitrin polypeptide) removes impurities that can reduce or remove anunwanted immune response, e.g., reducing cytokine activity.

In some embodiments, the polynucleotide of the invention (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) is purified prior to administration using columnchromatography (e.g., strong anion exchange HPLC, weak anion exchangeHPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC), or (LCMS)).

In some embodiments, the polynucleotide of the invention (e.g., apolynucleotide comprising a nucleotide sequence a Citrin polypeptide)purified using column chromatography (e.g., strong anion exchange HPLC,weak anion exchange HPLC, reverse phase HPLC (RP-HPLC, hydrophobicinteraction HPLC (HIC-HPLC), or (LCMS)) presents increased expression ofthe encoded Citrin protein compared to the expression level obtainedwith the same polynucleotide of the present disclosure purified by adifferent purification method.

In some embodiments, a column chromatography (e.g., strong anionexchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC),hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)) purifiedpolynucleotide comprises a nucleotide sequence encoding a Citrinpolypeptide comprising one or more of the point mutations known in theart.

In some embodiments, the use of RP-HPLC purified polynucleotideincreases Citrin protein expression levels in cells when introduced intothose cells, e.g., by 10-100%, i.e., at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 90%, at least about95%, or at least about 100% with respect to the expression levels ofCitrin protein in the cells before the RP-HPLC purified polynucleotidewas introduced in the cells, or after a non-RP-HPLC purifiedpolynucleotide was introduced in the cells.

In some embodiments, the use of RP-HPLC purified polynucleotideincreases functional Citrin protein expression levels in cells whenintroduced into those cells, e.g., by 10-100%, i.e., at least about 10%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 90%,at least about 95%, or at least about 100% with respect to thefunctional expression levels of Citrin protein in the cells before theRP-HPLC purified polynucleotide was introduced in the cells, or after anon-RP-HPLC purified polynucleotide was introduced in the cells.

In some embodiments, the use of RP-HPLC purified polynucleotideincreases detectable Citrin activity in cells when introduced into thosecells, e.g., by 10-100%, i.e., at least about 10%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 90%, at least about 95%,or at least about 100% with respect to the activity levels of functionalCitrin in the cells before the RP-HPLC purified polynucleotide wasintroduced in the cells, or after a non-RP-HPLC purified polynucleotidewas introduced in the cells.

In some embodiments, the purified polynucleotide is at least about 80%pure, at least about 85% pure, at least about 90% pure, at least about95% pure, at least about 96% pure, at least about 97% pure, at leastabout 98% pure, at least about 99% pure, or about 100% pure.

A quality assurance and/or quality control check can be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC. In another embodiment, the polynucleotide can besequenced by methods including, but not limited toreverse-transcriptase-PCR.

d. Quantification of Expressed Polynucleotides Encoding Citrin

In some embodiments, the polynucleotides of the present invention (e.g.,a polynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide), their expression products, as well as degradation productsand metabolites can be quantified according to methods known in the art.

In some embodiments, the polynucleotides of the present invention can bequantified in exosomes or when derived from one or more bodily fluid. Asused herein “bodily fluids” include peripheral blood, serum, plasma,ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow,synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid orpre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid,pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle,bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, andumbilical cord blood. Alternatively, exosomes can be retrieved from anorgan selected from the group consisting of lung, heart, pancreas,stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast,prostate, brain, esophagus, liver, and placenta.

In the exosome quantification method, a sample of not more than 2 mL isobtained from the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of a polynucleotide can bean expression level, presence, absence, truncation or alteration of theadministered construct. It is advantageous to correlate the level withone or more clinical phenotypes or with an assay for a human diseasebiomarker.

The assay can be performed using construct specific probes, cytometry,qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, massspectrometry, or combinations thereof while the exosomes can be isolatedusing immunohistochemical methods such as enzyme linked immunosorbentassay (ELISA) methods. Exosomes can also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides remaining or delivered. This ispossible because the polynucleotides of the present invention differfrom the endogenous forms due to the structural or chemicalmodifications.

In some embodiments, the polynucleotide can be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantifiedpolynucleotide can be analyzed in order to determine if thepolynucleotide can be of proper size, check that no degradation of thepolynucleotide has occurred. Degradation of the polynucleotide can bechecked by methods such as, but not limited to, agarose gelelectrophoresis, HPLC based purification methods such as, but notlimited to, strong anion exchange HPLC, weak anion exchange HPLC,reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillaryelectrophoresis (CE) and capillary gel electrophoresis (CGE).

21. Pharmaceutical Compositions and Formulations

The present invention provides pharmaceutical compositions andformulations that comprise any of the polynucleotides described above.In some embodiments, the composition or formulation further comprises adelivery agent.

In some embodiments, the composition or formulation can contain apolynucleotide comprising a sequence optimized nucleic acid sequencedisclosed herein which encodes a Citrin polypeptide. In someembodiments, the composition or formulation can contain a polynucleotide(e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF)having significant sequence identity to a sequence optimized nucleicacid sequence disclosed herein which encodes a Citrin polypeptide. Insome embodiments, the polynucleotide further comprises a miRNA bindingsite, e.g., a miRNA binding site that binds miR-126, miR-142, miR-144,miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27 andmiR-26a.

Pharmaceutical compositions or formulation can optionally comprise oneor more additional active substances, e.g., therapeutically and/orprophylactically active substances. Pharmaceutical compositions orformulation of the present invention can be sterile and/or pyrogen-free.General considerations in the formulation and/or manufacture ofpharmaceutical agents can be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety). Insome embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides to bedelivered as described herein.

Formulations and pharmaceutical compositions described herein can beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients, and then, if necessary and/or desirable,dividing, shaping and/or packaging the product into a desired single- ormulti-dose unit.

A pharmaceutical composition or formulation in accordance with thepresent disclosure can be prepared, packaged, and/or sold in bulk, as asingle unit dose, and/or as a plurality of single unit doses. As usedherein, a “unit dose” refers to a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure canvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered.

In some embodiments, the compositions and formulations described hereincan contain at least one polynucleotide of the invention. As anon-limiting example, the composition or formulation can contain 1, 2,3, 4 or 5 polynucleotides of the invention. In some embodiments, thecompositions or formulations described herein can comprise more than onetype of polynucleotide. In some embodiments, the composition orformulation can comprise a polynucleotide in linear and circular form.In another embodiment, the composition or formulation can comprise acircular polynucleotide and an in vitro transcribed (IVT)polynucleotide. In yet another embodiment, the composition orformulation can comprise an IVT polynucleotide, a chimericpolynucleotide and a circular polynucleotide.

Although the descriptions of pharmaceutical compositions andformulations provided herein are principally directed to pharmaceuticalcompositions and formulations that are suitable for administration tohumans, it will be understood by the skilled artisan that suchcompositions are generally suitable for administration to any otheranimal, e.g., to non-human animals, e.g. non-human mammals. The presentinvention provides pharmaceutical formulations that comprise apolynucleotide described herein (e.g., a polynucleotide comprising anucleotide sequence encoding a Citrin polypeptide). The polynucleotidesdescribed herein can be formulated using one or more excipients to: (1)increase stability; (2) increase cell transfection; (3) permit thesustained or delayed release (e.g., from a depot formulation of thepolynucleotide); (4) alter the biodistribution (e.g., target thepolynucleotide to specific tissues or cell types); (5) increase thetranslation of encoded protein in vivo; and/or (6) alter the releaseprofile of encoded protein in vivo. In some embodiments, thepharmaceutical formulation disclosed herein further comprises a deliveryagent comprising, e.g., a compound having the Formula (I), e.g., any ofCompounds 1-232, e.g., Compound 18; a compound having the Formula (III),(IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236;or a compound having the Formula (VIII), e.g., any of Compounds 419-428,e.g., Compound 428, or any combination thereof. In some embodiments, thedelivery agent comprises Compound 18, DSPC, Cholesterol, and Compound428, e.g., with a mole ratio of about 50:10:38.5:1.5.

A pharmaceutically acceptable excipient, as used herein, includes, butare not limited to, any and all solvents, dispersion media, or otherliquid vehicles, dispersion or suspension aids, diluents, granulatingand/or dispersing agents, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, binders, lubricants oroil, coloring, sweetening or flavoring agents, stabilizers,antioxidants, antimicrobial or antifungal agents, osmolality adjustingagents, pH adjusting agents, buffers, chelants, cryoprotectants, and/orbulking agents, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21st Edition, A. R. Gennaro(Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference in its entirety).

Exemplary diluents include, but are not limited to, calcium or sodiumcarbonate, calcium phosphate, calcium hydrogen phosphate, sodiumphosphate, lactose, sucrose, cellulose, microcrystalline cellulose,kaolin, mannitol, sorbitol, etc., and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, starches, pregelatinized starches, or microcrystallinestarch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone),(providone), cross-linked poly(vinyl-pyrrolidone) (crospovidone),cellulose, methylcellulose, carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), magnesium aluminum silicate(VEEGUM®), sodium lauryl sulfate, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g., acacia, agar, alginic acid,sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin),sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate[TWEEN®80], sorbitan monopalmitate [SPAN®40], glyceryl monooleate,polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g.,CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether[BRIJ®30]), PLUORINC®F 68, POLOXAMER®188, etc. and/or combinationsthereof.

Exemplary binding agents include, but are not limited to, starch,gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses,lactose, lactitol, mannitol), amino acids (e.g., glycine), natural andsynthetic gums (e.g., acacia, sodium alginate), ethylcellulose,hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., andcombinations thereof.

Oxidation is a potential degradation pathway for mRNA, especially forliquid mRNA formulations. In order to prevent oxidation, antioxidantscan be added to the formulations. Exemplary antioxidants include, butare not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate,benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine,butylated hydroxytoluene, monothioglycerol, sodium or potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc.,and combinations thereof.

Exemplary chelating agents include, but are not limited to,ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, fumaric acid, malic acid, phosphoric acid, sodiumedetate, tartaric acid, trisodium edetate, etc., and combinationsthereof.

Exemplary antimicrobial or antifungal agents include, but are notlimited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethyl paraben, propyl paraben, butyl paraben, benzoic acid,hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodiumsorbate, sodium propionate, sorbic acid, etc., and combinations thereof.

Exemplary preservatives include, but are not limited to, vitamin A,vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid,butylated hydroxyanisol, ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), etc., and combinations thereof.

In some embodiments, the pH of polynucleotide solutions are maintainedbetween pH 5 and pH 8 to improve stability. Exemplary buffers to controlpH can include, but are not limited to sodium phosphate, sodium citrate,sodium succinate, histidine (or histidine-HCl), sodium malate, sodiumcarbonate, etc., and/or combinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium or magnesium lauryl sulfate, etc., and combinations thereof.

The pharmaceutical composition or formulation described here can containa cryoprotectant to stabilize a polynucleotide described herein duringfreezing. Exemplary cryoprotectants include, but are not limited tomannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., andcombinations thereof.

The pharmaceutical composition or formulation described here can containa bulking agent in lyophilized polynucleotide formulations to yield a“pharmaceutically elegant” cake, stabilize the lyophilizedpolynucleotides during long term (e.g., 36 month) storage. Exemplarybulking agents of the present invention can include, but are not limitedto sucrose, trehalose, mannitol, glycine, lactose, raffinose, andcombinations thereof.

In some embodiments, the pharmaceutical composition or formulationfurther comprises a delivery agent. The delivery agent of the presentdisclosure can include, without limitation, liposomes, lipidnanoparticles, lipidoids, polymers, lipoplexes, microvesicles, exosomes,peptides, proteins, cells transfected with polynucleotides,hyaluronidase, nanoparticle mimics, nanotubes, conjugates, andcombinations thereof.

22. Delivery Agents

a. Lipid Compound

The present disclosure provides pharmaceutical compositions withadvantageous properties. The lipid compositions described herein may beadvantageously used in lipid nanoparticle compositions for the deliveryof therapeutic and/or prophylactic agents, e.g., mRNAs, to mammaliancells or organs. For example, the lipids described herein have little orno immunogenicity. For example, the lipid compounds disclosed hereinhave a lower immunogenicity as compared to a reference lipid (e.g., MC3,KC2, or DLinDMA). For example, a formulation comprising a lipiddisclosed herein and a therapeutic or prophylactic agent, e.g., mRNA,has an increased therapeutic index as compared to a correspondingformulation which comprises a reference lipid (e.g., MC3, KC2, orDLinDMA) and the same therapeutic or prophylactic agent.

In certain embodiments, the present application provides pharmaceuticalcompositions comprising:

-   -   (a) a polynucleotide comprising a nucleotide sequence encoding a        Citrin polypeptide; and    -   (b) a delivery agent.

In some embodiments, the delivery agent comprises a lipid compoundhaving the formula (I)

wherein

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR,        —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,        —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,        —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂,        —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(R)N(R)₂C(O)OR, and        each n is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H; R₈ is selected from the group consisting of C₃₋₆        carbocycle and heterocycle; R₉ is selected from the group        consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂,        C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.    -   In some embodiments, a subset of compounds of Formula (I)        includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n        is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H; each R is independently selected from the group        consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,    -   or salts or stereoisomers thereof, wherein alkyl and alkenyl        groups may be linear or branched.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In another embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to        14-membered heteroaryl having one or more heteroatoms selected        from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,        —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈,        —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,        —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,        —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂,        —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂,        —C(═NR₉)R, —C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl        having one or more heteroatoms selected from N, O, and S which        is substituted with one or more substituents selected from oxo        (═O), OH, amino, and C₁₋₃ alkyl, and each n is independently        selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; M and M′ are independently        selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,        —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,        —S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   R₈ is selected from the group consisting of C₃₋₆ carbocycle and        heterocycle;    -   R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆        alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle        and heterocycle;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In another embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to        14-membered heteroaryl having one or more heteroatoms selected        from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,        —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, and a 5- to        14-membered heterocycloalkyl having one or more heteroatoms        selected from N, O, and S which is substituted with one or more        substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl,        and each n is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In yet other embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to        14-membered heterocycle having one or more heteroatoms selected        from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,        —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈,        —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,        —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,        —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂,        —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR,        and —C(═NR₉)N(R)₂, and each n is independently selected from 1,        2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle        and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is        —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR, and        —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8-        to 14-membered heterocycloalkyl;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   R₈ is selected from the group consisting of C₃₋₆ carbocycle and        heterocycle;    -   R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆        alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle        and heterocycle;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In yet other embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to        14-membered heterocycle having one or more heteroatoms selected        from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,        —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, and each n is        independently selected from 1, 2, 3, 4, and 5; and when Q is a        5- to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which        n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1,        or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to        14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to        14-membered heteroaryl having one or more heteroatoms selected        from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,        —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈,        —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,        —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,        —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂,        —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR,        and —C(═NR₉)N(R)₂, and each n is independently selected from 1,        2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   R₈ is selected from the group consisting of C₃₋₆ carbocycle and        heterocycle;    -   R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆        alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle        and heterocycle;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to        14-membered heteroaryl having one or more heteroatoms selected        from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,        —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, and each n is        independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n is        selected from 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₁₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n is        selected from 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₁₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In still other embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂        and R₃, together with the atom to which they are attached, form        a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of —(CH₂)_(n)Q,        —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is        selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₁₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In still other embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂        and R₃, together with the atom to which they are attached, form        a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of —(CH₂)_(n)Q,        —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is        selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₁₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,        or salts or stereoisomers thereof.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IA):

or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5;    -   m is selected from 5, 6, 7, 8, and 9;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is        OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂,        —N(R)C(O)OR, heteroaryl or heterocycloalkyl;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a        heteroaryl group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA), or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6,        7, 8, and 9;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is        OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl        group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (II):

or a salt or stereoisomer thereof, wherein l is selected from 1, 2, 3,4, and 5;

-   -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2,        3, or 4, and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R,        —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,        —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a        heteroaryl group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (II), or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2,        3, or 4, and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl        group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, the compound of formula (I) is of the formula(IIa),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIb),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIc),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIe):

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (IIa), (IIb), (IIc), or(IIe) comprises an R₄ which is selected from —(CH₂)_(n)Q and—(CH₂)_(n)CHQR, wherein Q, R and n are as defined above.

In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle, wherein R is as defined above. In some aspects, n is 1 or2. In some embodiments, Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂.

In some embodiments, the compound of formula (I) is of the formula(IId),

or a salt thereof, wherein R₂ and R₃ are independently selected from thegroup consisting of C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl, n is selected from 2,3, and 4, and R′, R″, R₅, R₆ and m are as defined above.

In some aspects of the compound of formula (IId), R₂ is C₈ alkyl. Insome aspects of the compound of formula (IId), R₃ is C₅-C₉ alkyl. Insome aspects of the compound of formula (IId), m is 5, 7, or 9. In someaspects of the compound of formula (IId), each R₅ is H. In some aspectsof the compound of formula (IId), each R₆ is H.

In another aspect, the present application provides a lipid composition(e.g., a lipid nanoparticle (LNP)) comprising: (1) a compound having theformula (I); (2) optionally a helper lipid (e.g. a phospholipid); (3)optionally a structural lipid (e.g. a sterol); and (4) optionally alipid conjugate (e.g. a PEG-lipid). In exemplary embodiments, the lipidcomposition (e.g., LNP) further comprises a polynucleotide encoding aCitrin polypeptide, e.g., a polynucleotide encapsulated therein.

As used herein, the term “alkyl” or “alkyl group” means a linear orbranched, saturated hydrocarbon including one or more carbon atoms(e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms).

The notation “C₁₋₁₄ alkyl” means a linear or branched, saturatedhydrocarbon including 1-14 carbon atoms. An alkyl group can beoptionally substituted.

As used herein, the term “alkenyl” or “alkenyl group” means a linear orbranched hydrocarbon including two or more carbon atoms (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more carbon atoms) and at least one double bond.

The notation “C₂₋₁₄ alkenyl” means a linear or branched hydrocarbonincluding 2-14 carbon atoms and at least one double bond. An alkenylgroup can include one, two, three, four, or more double bonds. Forexample, C₁₈ alkenyl can include one or more double bonds. A C₁₈ alkenylgroup including two double bonds can be a linoleyl group. An alkenylgroup can be optionally substituted.

As used herein, the term “carbocycle” or “carbocyclic group” means amono- or multi-cyclic system including one or more rings of carbonatoms. Rings can be three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, or fifteen membered rings.

The notation “C₃₋₆ carbocycle” means a carbocycle including a singlering having 3-6 carbon atoms. Carbocycles can include one or more doublebonds and can be aromatic (e.g., aryl groups). Examples of carbocyclesinclude cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and1,2-dihydronaphthyl groups. Carbocycles can be optionally substituted.

As used herein, the term “heterocycle” or “heterocyclic group” means amono- or multi-cyclic system including one or more rings, where at leastone ring includes at least one heteroatom. Heteroatoms can be, forexample, nitrogen, oxygen, or sulfur atoms. Rings can be three, four,five, six, seven, eight, nine, ten, eleven, or twelve membered rings.Heterocycles can include one or more double bonds and can be aromatic(e.g., heteroaryl groups). Examples of heterocycles include imidazolyl,imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl,pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl,isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl,tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, andisoquinolyl groups. Heterocycles can be optionally substituted.

As used herein, a “biodegradable group” is a group that can facilitatefaster metabolism of a lipid in a subject. A biodegradable group can be,but is not limited to, —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, anaryl group, and a heteroaryl group.

As used herein, an “aryl group” is a carbocyclic group including one ormore aromatic rings. Examples of aryl groups include phenyl and naphthylgroups.

As used herein, a “heteroaryl group” is a heterocyclic group includingone or more aromatic rings. Examples of heteroaryl groups includepyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Botharyl and heteroaryl groups can be optionally substituted. For example, Mand M′ can be selected from the non-limiting group consisting ofoptionally substituted phenyl, oxazole, and thiazole. In the formulasherein, M and M′ can be independently selected from the list ofbiodegradable groups above.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groupscan be optionally substituted unless otherwise specified. Optionalsubstituents can be selected from the group consisting of, but are notlimited to, a halogen atom (e.g., a chloride, bromide, fluoride, oriodide group), a carboxylic acid (e.g., —C(O)OH), an alcohol (e.g., ahydroxyl, —OH), an ester (e.g., —C(O)OR or —OC(O)R), an aldehyde (e.g.,—C(O)H), a carbonyl (e.g., —C(O)R, alternatively represented by C═O), anacyl halide (e.g., —C(O)X, in which X is a halide selected from bromide,fluoride, chloride, and iodide), a carbonate (e.g., —OC(O)OR), an alkoxy(e.g., —OR), an acetal (e.g., —C(OR)₂R″″, in which each OR are alkoxygroups that can be the same or different and R″″ is an alkyl or alkenylgroup), a phosphate (e.g., P(O)₄ ³⁻), a thiol (e.g., —SH), a sulfoxide(e.g., —S(O)R), a sulfuric acid (e.g., —S(O)OH), a sulfonic acid (e.g.,—S(O)₂OH), a thial (e.g., —C(S)H), a sulfate (e.g., S(O)₄ ²⁻), asulfonyl (e.g., —S(O)₂—), an amide (e.g., —C(O)NR₂, or —N(R)C(O)R), anazido (e.g., —N₃), a nitro (e.g., —NO₂), a cyano (e.g., —CN), anisocyano (e.g., —NC), an acyloxy (e.g., —OC(O)R), an amino (e.g., —NR₂,—NRH, or —NH₂), a carbamoyl (e.g., —OC(O)NR₂, —OC(O)NRH, or —OC(O)NH₂),a sulfonamide (e.g., —S(O)₂NR₂, —S(O)₂NRH, —S(O)₂NH₂, —N(R)S(O)₂R,—N(H)S(O)₂R, —N(R)S(O)₂H, or —N(H)S(O)₂H), an alkyl group, an alkenylgroup, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group.

In any of the preceding, R is an alkyl or alkenyl group, as definedherein. In some embodiments, the substituent groups themselves can befurther substituted with, for example, one, two, three, four, five, orsix substituents as defined herein. For example, a C₁₋₆ alkyl group canbe further substituted with one, two, three, four, five, or sixsubstituents as described herein.

The compounds of any one of formulae (I), (IA), (II), (IIa), (IIb),(IIc), (IId), and (IIe) include one or more of the following featureswhen applicable.

In some embodiments, R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q isselected from a C₃₋₆ carbocycle, 5- to 14-membered aromatic ornon-aromatic heterocycle having one or more heteroatoms selected from N,O, S, and P, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n is independentlyselected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and a 5- to 14-membered heterocycloalkyl having one ormore heteroatoms selected from N, O, and S which is substituted with oneor more substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl,and each n is independently selected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heterocyclehaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5.

In another embodiment, R₄ is unsubstituted C₁₋₄ alkyl, e.g.,unsubstituted methyl.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is—N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is selected from the group consisting of—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q is —N(R)₂, andn is selected from 1, 2, 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₂ and R₃ are independently selected from the groupconsisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, orR₂ and R₃, together with the atom to which they are attached, form aheterocycle or carbocycle, and R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR,where Q is —N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, R₂ and R₃ are independently selected from thegroup consisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and—R*OR″, or R₂ and R₃, together with the atom to which they are attached,form a heterocycle or carbocycle.

In some embodiments, R₁ is selected from the group consisting of C₅₋₂₀alkyl and C₅₋₂₀ alkenyl.

In other embodiments, R₁ is selected from the group consisting of—R*YR″, —YR″, and —R″M′R′.

In certain embodiments, R₁ is selected from —R*YR″ and —YR″. In someembodiments, Y is a cyclopropyl group. In some embodiments, R* is C₈alkyl or C₈ alkenyl. In certain embodiments, R″ is C₃₋₁₂ alkyl. Forexample, R″ can be C₃ alkyl. For example, R″ can be C₄₋₈ alkyl (e.g.,C₄, C₅, C₆, C₇, or C₈ alkyl).

In some embodiments, R₁ is C₅₋₂₀ alkyl. In some embodiments, R₁ is C₆alkyl. In some embodiments, R₁ is C₈ alkyl. In other embodiments, R₁ isC₉ alkyl. In certain embodiments, R₁ is C₁₄ alkyl. In other embodiments,R₁ is C₁₈ alkyl.

In some embodiments, R₁ is C₅₋₂₀ alkenyl. In certain embodiments, R₁ isC₁₈ alkenyl. In some embodiments, R₁ is linoleyl.

In certain embodiments, R₁ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, orheptadeca-9-yl). In certain embodiments, R₁ is

In certain embodiments, R₁ is unsubstituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl. In certain embodiments, R′ is substituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl (e.g., substituted with a C₃₋₆ carbocycle such as1-cyclopropylnonyl).

In other embodiments, R₁ is —R″M′R′.

In some embodiments, R′ is selected from —R*YR″ and —YR″. In someembodiments, Y is C₃₋₈ cycloalkyl. In some embodiments, Y is C₆₋₁₀ aryl.In some embodiments, Y is a cyclopropyl group. In some embodiments, Y isa cyclohexyl group. In certain embodiments, R* is C₁ alkyl.

In some embodiments, R″ is selected from the group consisting of C₃₋₁₂alkyl and C₃₋₁₂ alkenyl. In some embodiments, R″ adjacent to Y is C₁alkyl. In some embodiments, R″ adjacent to Y is C₄₋₉ alkyl (e.g., C₄,C₅, C₆, C₇ or C₈ or C₉ alkyl).

In some embodiments, R′ is selected from C₄ alkyl and C₄ alkenyl. Incertain embodiments, R′ is selected from C₅ alkyl and C₅ alkenyl. Insome embodiments, R′ is selected from C₆ alkyl and C₆ alkenyl. In someembodiments, R′ is selected from C₇ alkyl and C₇ alkenyl. In someembodiments, R′ is selected from C₉ alkyl and C₉ alkenyl.

In other embodiments, R′ is selected from C₁₁ alkyl and C₁₁ alkenyl. Inother embodiments, R′ is selected from C₁₂ alkyl, C₁₂ alkenyl, C₁₃alkyl, C₁₃ alkenyl, C₁₄ alkyl, C₁₄ alkenyl, C₁₅ alkyl, C₁₅ alkenyl, C₁₆alkyl, C₁₆ alkenyl, C₁₇ alkyl, C₁₇ alkenyl, C₁₅ alkyl, and C₁₅ alkenyl.In certain embodiments, R′ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl orheptadeca-9-yl). In certain embodiments, R′ is

In certain embodiments, R′ is unsubstituted C₁₋₁₈ alkyl. In certainembodiments, R′ is substituted C₁₋₁₈ alkyl (e.g., C₁₋₁₅ alkylsubstituted with a C₃₋₆ carbocycle such as 1-cyclopropylnonyl).

In some embodiments, R″ is selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl. In some embodiments, R″ is C₃ alkyl, C₄ alkyl,C₅ alkyl, C₆ alkyl, C₇ alkyl, or C₈ alkyl. In some embodiments, R″ is C₉alkyl, C₁₀ alkyl, C₁₁ alkyl, C₁₂ alkyl, C₁₃ alkyl, or C₁₄ alkyl.

In some embodiments, M′ is —C(O)O—. In some embodiments, M′ is —OC(O)—.

In other embodiments, M′ is an aryl group or heteroaryl group. Forexample, M′ can be selected from the group consisting of phenyl,oxazole, and thiazole.

In some embodiments, M is —C(O)O— In some embodiments, M is —OC(O)—. Insome embodiments, M is —C(O)N(R′)—. In some embodiments, M is—P(O)(OR′)O—.

In other embodiments, M is an aryl group or heteroaryl group. Forexample, M can be selected from the group consisting of phenyl, oxazole,and thiazole.

In some embodiments, M is the same as M′. In other embodiments, M isdifferent from M′.

In some embodiments, each R₅ is H. In certain such embodiments, each R₆is also H.

In some embodiments, R₇ is H. In other embodiments, R₇ is C₁₋₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In some embodiments, R₂ and R₃ are independently C₅₋₁₄ alkyl or C₅₋₁₄alkenyl.

In some embodiments, R₂ and R₃ are the same. In some embodiments, R₂ andR₃ are C₈ alkyl. In certain embodiments, R₂ and R₃ are C₂ alkyl. Inother embodiments, R₂ and R₃ are C₃ alkyl. In some embodiments, R₂ andR₃ are C₄ alkyl. In certain embodiments, R₂ and R₃ are C₅ alkyl. Inother embodiments, R₂ and R₃ are C₆ alkyl. In some embodiments, R₂ andR₃ are C₇ alkyl.

In other embodiments, R₂ and R₃ are different. In certain embodiments,R₂ is C₈ alkyl. In some embodiments, R₃ is C₁₋₇ (e.g., C₁, C₂, C₃, C₄,C₅, C₆, or C₇ alkyl) or C₉ alkyl.

In some embodiments, R₇ and R₃ are H.

In certain embodiments, R₂ is H.

In some embodiments, m is 5, 7, or 9.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.

In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R),—C(R)N(R)₂C(O)OR, a carbocycle, and a heterocycle.

In certain embodiments, Q is —OH.

In certain embodiments, Q is a substituted or unsubstituted 5- to10-membered heteroaryl, e.g., Q is an imidazole, a pyrimidine, a purine,2-amino-1,9-dihydro-6H-purin-6-one-9-yl (or guanin-9-yl), adenin-9-yl,cytosin-1-yl, or uracil-1-yl. In certain embodiments, Q is a substituted5- to 14-membered heterocycloalkyl, e.g., substituted with one or moresubstituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl. Forexample, Q is 4-methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, orisoindolin-2-yl-1,3-dione.

In certain embodiments, Q is an unsubstituted or substituted C₆₋₁₀ aryl(such as phenyl) or C₃₋₆ cycloalkyl.

In some embodiments, n is 1. In other embodiments, n is 2. In furtherembodiments, n is 3. In certain other embodiments, n is 4. For example,R₄ can be —(CH₂)₂OH. For example, R₄ can be —(CH₂)₃OH. For example, R₄can be —(CH₂)₄OH. For example, R₄ can be benzyl. For example, R₄ can be4-methoxybenzyl.

In some embodiments, R₄ is a C₃₋₆ carbocycle. In some embodiments, R₄ isa C₃₋₆ cycloalkyl. For example, R₄ can be cyclohexyl optionallysubstituted with e.g., OH, halo, C₁₋₆ alkyl, etc. For example, R₄ can be2-hydroxycyclohexyl.

In some embodiments, R is H.

In some embodiments, R is unsubstituted C₁₋₃ alkyl or unsubstituted C₂₋₃alkenyl. For example, R₄ can be —CH₂CH(OH)CH₃ or —CH₂CH(OH)CH₂CH₃.

In some embodiments, R is substituted C₁₋₃ alkyl, e.g., CH₂OH. Forexample, R₄ can be —CH₂CH(OH)CH₂OH.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a 5- to14-membered aromatic or non-aromatic heterocycle having one or moreheteroatoms selected from N, O, S, and P. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form anoptionally substituted C₃₋₂₀ carbocycle (e.g., C₃₋₁₈ carbocycle, C₃₋₁₅carbocycle, C₃₋₁₂ carbocycle, or C₃₋₁₀ carbocycle), either aromatic ornon-aromatic. In some embodiments, R₂ and R₃, together with the atom towhich they are attached, form a C₃₋₆ carbocycle. In other embodiments,R₂ and R₃, together with the atom to which they are attached, form a C₆carbocycle, such as a cyclohexyl or phenyl group. In certainembodiments, the heterocycle or C₃₋₆ carbocycle is substituted with oneor more alkyl groups (e.g., at the same ring atom or at adjacent ornon-adjacent ring atoms). For example, R₂ and R₃, together with the atomto which they are attached, can form a cyclohexyl or phenyl groupbearing one or more C₅ alkyl substitutions. In certain embodiments, theheterocycle or C₃₋₆ carbocycle formed by R₂ and R₃, is substituted witha carbocycle groups. For example, R₂ and R₃, together with the atom towhich they are attached, can form a cyclohexyl or phenyl group that issubstituted with cyclohexyl. In some embodiments, R₂ and R₃, togetherwith the atom to which they are attached, form a C₇₋₁₅ carbocycle, suchas a cycloheptyl, cyclopentadecanyl, or naphthyl group.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle. In other embodiments, Q is selected from the groupconsisting of an imidazole, a pyrimidine, and a purine.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a C₃₋₆carbocycle, such as a phenyl group. In certain embodiments, theheterocycle or C₃₋₆ carbocycle is substituted with one or more alkylgroups (e.g., at the same ring atom or at adjacent or non-adjacent ringatoms). For example, R₂ and R₃, together with the atom to which they areattached, can form a phenyl group bearing one or more C₅ alkylsubstitutions.

In some embodiments, the pharmaceutical compositions of the presentdisclosure, the compound of formula (I) is selected from the groupconsisting of:

and salts or stereoisomers thereof.

In other embodiments, the compound of Formula (I) is selected from thegroup consisting of Compound 1-Compound 147, or salt or stereoisomersthereof.

In some embodiments ionizable lipids including a central piperazinemoiety are provided.

In some embodiments, the delivery agent comprises a lipid compoundhaving the formula (III)

or salts or stereoisomers thereof, whereinring A is

t is 1 or 2;

-   -   A₁ and A₂ are each independently selected from CH or N;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   R₁, R₂, R₃, R₄, and R₅ are independently selected from the group        consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″,        and —R*OR″;    -   each M is independently selected from the group consisting        of —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—,        —C(S)—, —C(S)S—, —S C(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an        aryl group, and a heteroaryl group;    -   X¹, X², and X³ are independently selected from the group        consisting of a bond, —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—,        —C(O)O—, —OC(O)—, —C(O)—CH₂—, —CH₂—C(O)—, —C(O)O—CH₂—,        —OC(O)—CH₂—, —CH₂—C(O)O—, —CH₂—OC(O)—, —CH(OH)—, —C(S)—, and        —CH(SH)—;    -   each Y is independently a C₃₋₆ carbocycle;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl and a C₃₋₆ carbocycle;    -   each R′ is independently selected from the group consisting of        C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, and H; and    -   each R″ is independently selected from the group consisting of        C₃₋₁₂ alkyl and C₃₋₁₂ alkenyl,        wherein when ring A is

then

-   -   i) at least one of X¹, X², and X³ is not —CH₂—; and/or    -   ii) at least one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.

In some embodiments, the compound is of any of formulae (IIIa1)-(IIIa6):

The compounds of Formula (III) or any of (IIIa1)-(IIIa6) include one ormore of the following features when applicable.

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A

In some embodiments, ring A is

wherein ring, in which the N atom is connected with X².

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is CH.

In some embodiments, A₁ is N and A₂ is CH.

In some embodiments, A₁ is CH and A₂ is N.

In some embodiments, at least one of X¹, X², and X³ is not —CH₂—. Forexample, in certain embodiments, X¹ is not —CH₂—. In some embodiments,at least one of X¹, X², and X³ is —C(O)—.

In some embodiments, X² is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—.

In some embodiments, X³ is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—. Inother embodiments, X³ is —CH₂—.

In some embodiments, X³ is a bond or —(CH₂)₂—.

In some embodiments, R₁ and R₂ are the same. In certain embodiments, R₁,R₂, and R₃ are the same. In some embodiments, R₄ and R₅ are the same. Incertain embodiments, R₁, R₂, R₃, R₄, and R₅ are the same.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.In some embodiments, at most one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.For example, at least one of R₁, R₂, and R₃ may be —R″MR′, and/or atleast one of R₄ and R₅ is —R″MR′. In certain embodiments, at least one Mis —C(O)O—. In some embodiments, each M is —C(O)O—. In some embodiments,at least one M is —OC(O)—. In some embodiments, each M is —OC(O)—. Insome embodiments, at least one M is —OC(O)O—. In some embodiments, eachM is —OC(O)O—. In some embodiments, at least one R″ is C₃ alkyl. Incertain embodiments, each R″ is C₃ alkyl. In some embodiments, at leastone R″ is C₅ alkyl. In certain embodiments, each R″ is C₅ alkyl. In someembodiments, at least one R″ is C₆ alkyl. In certain embodiments, eachR″ is C₆ alkyl. In some embodiments, at least one R″ is C₇ alkyl. Incertain embodiments, each R″ is C₇ alkyl. In some embodiments, at leastone R′ is C₅ alkyl. In certain embodiments, each R′ is C₅ alkyl. Inother embodiments, at least one R′ is C₁ alkyl. In certain embodiments,each R′ is C₁ alkyl. In some embodiments, at least one R′ is C₂ alkyl.In certain embodiments, each R′ is C₂ alkyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is C₁₂alkyl. In certain embodiments, each of R₁, R₂, R₃, R₄, and R₅ are C₁₂alkyl.

In certain embodiments, the compound is selected from the groupconsisting of:

In some embodiments, the delivery agent comprises Compound 236.

In some embodiments, the delivery agent comprises a compound having theformula (IV)

or salts or stereoisomer thereof, wherein

-   -   A₁ and A₂ are each independently selected from CH or N and at        least one of A₁ and A₂ is N;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   R₁, R₂, R₃, R₄, and R₅ are independently selected from the group        consisting of C₆₋₂₀ alkyl and C₆₋₂₀ alkenyl;        wherein when ring A is

then

-   -   i) R₁, R₂, R₃, R₄, and R₅ are the same, wherein R₁ is not C₁₂        alkyl, C₁₈ alkyl, or C₁₈ alkenyl;    -   ii) only one of R₁, R₂, R₃, R₄, and R₅ is selected from C₆₋₂₀        alkenyl;    -   iii) at least one of R₁, R₂, R₃, R₄, and R₅ have a different        number of carbon atoms than at least one other of R₁, R₂, R₃,        R₄, and R₅;    -   iv) R₁, R₂, and R₃ are selected from C₆₋₂₀ alkenyl, and R₄ and        R₅ are selected from C₆₋₂₀ alkyl; or    -   v) R₁, R₂, and R₃ are selected from C₆₋₂₀ alkyl, and R₄ and R₅        are selected from C₆₋₂₀ alkenyl.

In some embodiments, the compound is of formula (IVa):

The compounds of Formula (IV) or (IVa) include one or more of thefollowing features when applicable.

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is CH.

In some embodiments, A₁ is N and A₂ is CH.

In some embodiments, A₁ is CH and A₂ is N.

In some embodiments, R₁, R₂, R₃, R₄, and R₅ are the same, and are notC₁₂ alkyl, C₁₈ alkyl, or C₁₈ alkenyl. In some embodiments, R₁, R₂, R₃,R₄, and R₅ are the same and are C₉ alkyl or C₁₄ alkyl.

In some embodiments, only one of R₁, R₂, R₃, R₄, and R₅ is selected fromC₆₋₂₀ alkenyl. In certain such embodiments, R₁, R₂, R₃, R₄, and R₅ havethe same number of carbon atoms. In some embodiments, R₄ is selectedfrom C₅₋₂₀ alkenyl. For example, R₄ may be C₁₂ alkenyl or C₁₈ alkenyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ have adifferent number of carbon atoms than at least one other of R₁, R₂, R₃,R₄, and R₅.

In certain embodiments, R₁, R₂, and R₃ are selected from C₆₋₂₀ alkenyl,and R₄ and R₅ are selected from C₆₋₂₀ alkyl. In other embodiments, R₁,R₂, and R₃ are selected from C₆₋₂₀ alkyl, and R₄ and R₅ are selectedfrom C₆₋₂₀ alkenyl. In some embodiments, R₁, R₂, and R₃ have the samenumber of carbon atoms, and/or R₄ and R₅ have the same number of carbonatoms. For example, R₁, R₂, and R₃, or R₄ and R₅, may have 6, 8, 9, 12,14, or 18 carbon atoms. In some embodiments, R₁, R₂, and R₃, or R₄ andR₅, are C₁₈ alkenyl (e.g., linoleyl). In some embodiments, R₁, R₂, andR₃, or R₄ and R₅, are alkyl groups including 6, 8, 9, 12, or 14 carbonatoms.

In some embodiments, R₁ has a different number of carbon atoms than R₂,R₃, R₄, and R₅. In other embodiments, R₃ has a different number ofcarbon atoms than R₁, R₂, R₄, and R₅. In further embodiments, R₄ has adifferent number of carbon atoms than R₁, R₂, R₃, and R₅.

In some embodiments, the compound is selected from the group consistingof:

In other embodiments, the delivery agent comprises a compound having theformula (V)

or salts or stereoisomers thereof, in which

-   -   A₃ is CH or N;    -   A₄ is CH₂ or NH; and at least one of A₃ and A₄ is N or NH;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   R₁, R₂, and R₃ are independently selected from the group        consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″,        and —R*OR″;    -   each M is independently selected        from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—,        —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl        group, and a heteroaryl group;    -   X¹ and X² are independently selected from the group consisting        of —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—,        —C(O)—CH₂—, —CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—,        —CH₂—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—;    -   each Y is independently a C₃₋₆ carbocycle;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl and a C₃₋₆ carbocycle;    -   each R′ is independently selected from the group consisting of        C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, and H; and    -   each R″ is independently selected from the group consisting of        C₃₋₁₂ alkyl and C₃₋₁₂ alkenyl.

In some embodiments, the compound is of formula (Va):

The compounds of Formula (V) or (Va) include one or more of thefollowing features when applicable.

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₃ and A₄ is N or NH.

In some embodiments, A₃ is N and A₄ is NH.

In some embodiments, A₃ is N and A₄ is CH₂.

In some embodiments, A₃ is CH and A₄ is NH.

In some embodiments, at least one of X¹ and X² is not —CH₂—. Forexample, in certain embodiments, X¹ is not —CH₂—. In some embodiments,at least one of X¹ and X² is —C(O)—.

In some embodiments, X² is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—.

In some embodiments, R₁, R₂, and R₃ are independently selected from thegroup consisting of C₅₋₂₀ alkyl and C₅₋₂₀ alkenyl. In some embodiments,R₁, R₂, and R₃ are the same. In certain embodiments, R₁, R₂, and R₃ areC₆, C₉, C₁₂, or C₁₄ alkyl. In other embodiments, R₁, R₂, and R₃ are C₁₈alkenyl. For example, R₁, R₂, and R₃ may be linoleyl.

In some embodiments, the compound is selected from the group consistingof:

In other embodiments, the delivery agent comprises a compound having theformula (VI):

or salts or stereoisomers thereof, in which

-   -   A₆ and A₇ are each independently selected from CH or N, wherein        at least one of A₆ and A₇ is N;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   X⁴ and X⁵ are independently selected from the group consisting        of —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—,        —C(O)—CH₂—, —CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—,        —CH₂—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—;    -   R₁, R₂, R₃, R₄, and R₅ each are independently selected from the        group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″,        —YR″, and —R*OR″;    -   each M is independently selected from the group consisting        of —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—,        —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl        group, and a heteroaryl group;    -   each Y is independently a C₃₋₆ carbocycle;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl and a C₃₋₆ carbocycle;    -   each R′ is independently selected from the group consisting of        C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, and H; and    -   each R″ is independently selected from the group consisting of        C₃₋₁₂ alkyl and C₃₋₁₂ alkenyl.

In some embodiments, R₁, R₂, R₃, R₄, and R₅ each are independentlyselected from the group consisting of C₆₋₂₀ alkyl and C₆₋₂₀ alkenyl.

In some embodiments, R₁ and R₂ are the same. In certain embodiments, R₁,R₂, and R₃ are the same. In some embodiments, R₄ and R₅ are the same. Incertain embodiments, R₁, R₂, R₃, R₄, and R₅ are the same.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is C₉₋₁₂alkyl. In certain embodiments, each of R₁, R₂, R₃, R₄, and R₅independently is C₉, C₁₂ or C₁₄ alkyl. In certain embodiments, each ofR₁, R₂, R₃, R₄, and R₅ is C₉ alkyl.

In some embodiments, A₆ is N and A₇ is N. In some embodiments, A₆ is CHand A₇ is N.

In some embodiments, X⁴ is —CH₂— and X⁵ is —C(O)—. In some embodiments,X⁴ and X⁵ are —C(O)—.

In some embodiments, when A₆ is N and A₇ is N, at least one of X⁴ and X⁵is not —CH₂—, e.g., at least one of X⁴ and X⁵ is —C(O)—. In someembodiments, when A₆ is N and A₇ is N, at least one of R₁, R₂, R₃, R₄,and R₅ is —R″MR′.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is not—R″MR′.

In some embodiments, the compound is

In other embodiments, the delivery agent comprises a compound having theformula:

Amine moieties of the lipid compounds disclosed herein can be protonatedunder certain conditions. For example, the central amine moiety of alipid according to formula (I) is typically protonated (i.e., positivelycharged) at a pH below the pKa of the amino moiety and is substantiallynot charged at a pH above the pKa. Such lipids can be referred toionizable amino lipids.

In one specific embodiment, the ionizable amino lipid is Compound 18. Inanother embodiment, the ionizable amino lipid is Compound 236.

In some embodiments, the amount the ionizable amino lipid, e.g.,compound of formula (I) ranges from about 1 mol % to 99 mol % in thelipid composition.

In one embodiment, the amount of the ionizable amino lipid, e.g.,compound of formula (I), is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99 mol % in the lipid composition.

In one embodiment, the amount of the ionizable amino lipid, e.g., thecompound of formula (I), ranges from about 30 mol % to about 70 mol %,from about 35 mol % to about 65 mol %, from about 40 mol % to about 60mol %, and from about 45 mol % to about 55 mol % in the lipidcomposition.

In one specific embodiment, the amount of the ionizable amino lipid,e.g., compound of formula (I), is about 50 mol % in the lipidcomposition.

In addition to the ionizable amino lipid disclosed herein, e.g.,compound of formula (I), the lipid composition of the pharmaceuticalcompositions disclosed herein can comprise additional components such asphospholipids, structural lipids, PEG-lipids, and any combinationthereof.

b. Additional Components in the Lipid Composition

(i) Phospholipids

The lipid composition of the pharmaceutical composition disclosed hereincan comprise one or more phospholipids, for example, one or moresaturated or (poly)unsaturated phospholipids or a combination thereof.In general, phospholipids comprise a phospholipid moiety and one or morefatty acid moieties.

A phospholipid moiety can be selected, for example, from thenon-limiting group consisting of phosphatidyl choline, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidicacid, 2-lysophosphatidyl choline, and a sphingomyelin.

A fatty acid moiety can be selected, for example, from the non-limitinggroup consisting of lauric acid, myristic acid, myristoleic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleicacid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid,arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoicacid, and docosahexaenoic acid.

Particular phospholipids can facilitate fusion to a membrane. Forexample, a cationic phospholipid can interact with one or morenegatively charged phospholipids of a membrane (e.g., a cellular orintracellular membrane). Fusion of a phospholipid to a membrane canallow one or more elements (e.g., a therapeutic agent) of alipid-containing composition (e.g., LNPs) to pass through the membranepermitting, e.g., delivery of the one or more elements to a targettissue.

Non-natural phospholipid species including natural species withmodifications and substitutions including branching, oxidation,cyclization, and alkynes are also contemplated. For example, aphospholipid can be functionalized with or cross-linked to one or morealkynes (e.g., an alkenyl group in which one or more double bonds isreplaced with a triple bond). Under appropriate reaction conditions, analkyne group can undergo a copper-catalyzed cycloaddition upon exposureto an azide. Such reactions can be useful in functionalizing a lipidbilayer of a nanoparticle composition to facilitate membrane permeationor cellular recognition or in conjugating a nanoparticle composition toa useful component such as a targeting or imaging moiety (e.g., a dye).

Phospholipids include, but are not limited to, glycerophospholipids suchas phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.Phospholipids also include phosphosphingolipid, such as sphingomyelin.

Examples of phospholipids include, but are not limited to, thefollowing:

In certain embodiments, a phospholipid useful or potentially useful inthe present invention is an analog or variant of DSPC. In certainembodiments, a phospholipid useful or potentially useful in the presentinvention is a compound of Formula (IX):

or a salt thereof, wherein:

each R¹ is independently optionally substituted alkyl; or optionally twoR¹ are joined together with the intervening atoms to form optionallysubstituted monocyclic carbocyclyl or optionally substituted monocyclicheterocyclyl; or optionally three R¹ are joined together with theintervening atoms to form optionally substituted bicyclic carbocyclyl oroptionally substitute bicyclic heterocyclyl;

-   -   n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   A is of the formula:

-   -   each instance of L² is independently a bond or optionally        substituted C₁₋₆ alkylene, wherein one methylene unit of the        optionally substituted C₁₋₆ alkylene is optionally replaced with        —O—, —N(R^(N))—, —S—, —C(O)—, —C(O)N(R^(N))—, —NR^(N)C(O)—,        —C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—, or        —NR^(N)C(O)N(RN)—;    -   each instance of R² is independently optionally substituted        C₁₋₃₀ alkyl, optionally substituted C₁₋₃₀ alkenyl, or optionally        substituted C₁₋₃₀ alkynyl; optionally wherein one or more        methylene units of R² are independently replaced with optionally        substituted carbocyclylene, optionally substituted        heterocyclylene, optionally substituted arylene, optionally        substituted heteroarylene, —N(R^(N))—, —O—, —S—, —C(O)—,        —C(O)N(R^(N))—, —NR^(N)C(O)—, —NR^(N)C(O)N(R^(N))—, —C(O)O—,        —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—, —C(O)S—,        —SC(O)—, —C(═NR^(N))—, —C(═NR^(N))N(R^(N))—, —NR^(N)C(═NR^(N))—,        —NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—, —C(S)N(R^(N))—,        —NR^(N)C(S)—, —NR^(N)C(S)N(R^(N))—, —S(O)—, —OS(O)—, —S(O)O—,        —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—, —N(R^(N))S(O)—,        —S(O)N(R^(N))—, —N(R^(N))S(O)N(R^(N))—, —OS(O)N(R^(N))—,        —N(R^(N))S(O)O—, —S(O)₂—, —N(R^(N))S(O)₂—, —S(O)₂N(R^(N))—,        —N(R^(N))S(O)₂N(R^(N))—, —OS(O)₂N(R^(N))—, or —N(R^(N))S(O)₂O—;    -   each instance of R^(N) is independently hydrogen, optionally        substituted alkyl, or a nitrogen protecting group;    -   Ring B is optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl, or        optionally substituted heteroaryl; and    -   p is 1 or 2;    -   provided that the compound is not of the formula:

wherein each instance of R² is independently unsubstituted alkyl,unsubstituted alkenyl, or unsubstituted alkynyl.

Phospholipid Head Modifications

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified phospholipid head (e.g., amodified choline group). In certain embodiments, a phospholipid with amodified head is DSPC, or analog thereof, with a modified quaternaryamine. For example, in embodiments of Formula (IX), at least one of R¹is not methyl. In certain embodiments, at least one of R¹ is nothydrogen or methyl. In certain embodiments, the compound of Formula (IX)is of one of the following formulae:

or a salt thereof, wherein:

-   -   each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   each v is independently 1, 2, or 3.

In certain embodiments, the compound of Formula (IX) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of thefollowing:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is of Formula (IX-a):

or a salt thereof.

In certain embodiments, phospholipids useful or potentially useful inthe present invention comprise a modified core. In certain embodiments,a phospholipid with a modified core described herein is DSPC, or analogthereof, with a modified core structure. For example, in certainembodiments of Formula (IX-a), group A is not of the following formula:

In certain embodiments, the compound of Formula (IX-a) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of thefollowing:

or salts thereof.

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a cyclic moiety in place of theglyceride moiety. In certain embodiments, a phospholipid useful in thepresent invention is DSPC, or analog thereof, with a cyclic moiety inplace of the glyceride moiety. In certain embodiments, the compound ofFormula (IX) is of Formula (IX-b):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-1):

or a salt thereof, wherein:

-   -   w is 0, 1, 2, or 3.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-2):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-3):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-4):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is one of thefollowing:

or salts thereof

Phospholipid Tail Modifications

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified tail. In certain embodiments,a phospholipid useful or potentially useful in the present invention isDSPC, or analog thereof, with a modified tail. As described herein, a“modified tail” may be a tail with shorter or longer aliphatic chains,aliphatic chains with branching introduced, aliphatic chains withsubstituents introduced, aliphatic chains wherein one or more methylenesare replaced by cyclic or heteroatom groups, or any combination thereof.For example, in certain embodiments, the compound of (IX) is of Formula(IX-a), or a salt thereof, wherein at least one instance of R² is eachinstance of R² is optionally substituted C₁₋₃₀ alkyl, wherein one ormore methylene units of R² are independently replaced with optionallysubstituted carbocyclylene, optionally substituted heterocyclylene,optionally substituted arylene, optionally substituted heteroarylene,—N(R^(N))—, —O—, —S—, —C(O)—, —C(O)N(R^(N))—, —NR^(N)C(O)—,—NR^(N)C(O)N(R^(N))—, —C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—,—NR^(N)C(O)O—, —C(O)S—, —SC(O)—, —C(═NR^(N))—, —C(═NR^(N))N(R^(N))—,—NR^(N)C(═NR^(N))—, —NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—, —C(S)N(R^(N))—,—NR^(N)C(S)—, —NR^(N)C(S)N(R^(N))—, —S(O)—, —OS(O)—, —S(O)—, —OS(O)O—,—OS(O)₂—, —S(O)₂O—, —OS(O)₂O—, —N(R^(N))S(O)—, —S(O)N(R^(N))—,—N(R^(N))S(O)N(R^(N))—, —OS(O)N(R^(N))—, —N(R^(N))S(O)O—, —S(O)₂—,—N(R^(N))S(O)₂—, —S(O)₂N(R^(N))—, —N(R^(N))S(O)₂N(R^(N))—,—OS(O)₂N(R^(N))—, or —N(R^(N))S(O)₂O—.

In certain embodiments, the compound of Formula (IX) is of Formula(IX-c):

or a salt thereof, wherein:

-   -   each x is independently an integer between 0-30, inclusive; and    -   each instance is G is independently selected from the group        consisting of optionally substituted carbocyclylene, optionally        substituted heterocyclylene, optionally substituted arylene,        optionally substituted heteroarylene, —N(R^(N))—, —O—, —S—,        —C(O)—, —C(O)N(R^(N))—, —NR^(N)C(O)—, —NR^(N)C(O)N(R^(N))—,        —C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—,        —C(O)S—, —SC(O)—, —C(═NR^(N))—, —C(═NR^(N))N(R^(N))—,        —NR^(N)C(═NR^(N))—, —NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—,        —C(S)N(R^(N))—, —NR^(N)C(S)—, —NR^(N)C(S)N(R^(N))—, —S(O)—,        —OS(O)—, —S(O)O—, —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—,        —N(R^(N))S(O)—, —S(O)N(R^(N))—, —N(R^(N))S(O)N(R^(N))—,        —OS(O)N(R^(N))—, —N(R^(N))S(O)O—, —S(O)₂—, —N(R^(N))S(O)₂—,        —S(O)₂N(R^(N))—, —N(R^(N))S(O)₂N(R^(N))—, —OS(O)₂N(R^(N))—, or        —N(R^(N))S(O)₂O—. Each possibility represents a separate        embodiment of the present invention.

In certain embodiments, the compound of Formula (IX-c) is of Formula(IX-c-1):

or salt thereof, wherein:

-   -   each instance of v is independently 1, 2, or 3.

In certain embodiments, the compound of Formula (IX-c) is of Formula(IX-c-2):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is of thefollowing formula:

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is the following:

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is of Formula(IX-c-3):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is of thefollowing formulae:

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is the following:

or a salt thereof.

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified phosphocholine moiety,wherein the alkyl chain linking the quaternary amine to the phosphorylgroup is not ethylene (e.g., n is not 2). Therefore, in certainembodiments, a phospholipid useful or potentially useful in the presentinvention is a compound of Formula (IX), wherein n is 1, 3, 4, 5, 6, 7,8, 9, or 10. For example, in certain embodiments, a compound of Formula(IX) is of one of the following formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of thefollowing:

or salts thereof.

(ii) Alternative Lipids

In certain embodiments, an alternative lipid is used in place of aphospholipid of the invention. Non-limiting examples of such alternativelipids include the following:

(iii) Structural Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more structural lipids. As used herein, the term“structural lipid” refers to sterols and also to lipids containingsterol moieties.

Incorporation of structural lipids in the lipid nanoparticle may helpmitigate aggregation of other lipids in the particle. Structural lipidscan be selected from the group including but not limited to,cholesterol, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid,alpha-tocopherol, hopanoids, phytosterols, steroids, and mixturesthereof. In some embodiments, the structural lipid is a sterol. Asdefined herein, “sterols” are a subgroup of steroids consisting ofsteroid alcohols. In certain embodiments, the structural lipid is asteroid. In certain embodiments, the structural lipid is cholesterol. Incertain embodiments, the structural lipid is an analog of cholesterol.In certain embodiments, the structural lipid is alpha-tocopherol.Examples of structural lipids include, but are not limited to, thefollowing:

In one embodiment, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition of a pharmaceuticalcomposition disclosed herein ranges from about 20 mol % to about 60 mol%, from about 25 mol % to about 55 mol %, from about 30 mol % to about50 mol %, or from about 35 mol % to about 45 mol %.

In one embodiment, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition disclosed herein rangesfrom about 25 mol % to about 30 mol %, from about 30 mol % to about 35mol %, or from about 35 mol % to about 40 mol %.

In one embodiment, the amount of the structural lipid (e.g., a sterolsuch as cholesterol) in the lipid composition disclosed herein is about24 mol %, about 29 mol %, about 34 mol %, or about 39 mol %.

In some embodiments, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition disclosed herein is atleast about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, or 60 mol %.

(iv) Polyethylene Glycol (PEG)-Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more a polyethylene glycol (PEG) lipid.

As used herein, the term “PEG-lipid” refers to polyethylene glycol(PEG)-modified lipids. Non-limiting examples of PEG-lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modifieddialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipidsare also referred to as PEGylated lipids. For example, a PEG lipid canbe PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPElipid.

In some embodiments, the PEG-lipid includes, but not limited to1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl,PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG),PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

In one embodiment, the PEG-lipid is selected from the group consistingof a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidicacid, a PEG-modified ceramide, a PEG-modified dialkylamine, aPEG-modified diacylglycerol, a PEG-modified dialkylglycerol, andmixtures thereof.

In some embodiments, the lipid moiety of the PEG-lipids includes thosehaving lengths of from about C₁₄ to about C₂₂, preferably from about C₁₄to about C₁₆. In some embodiments, a PEG moiety, for example anmPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000daltons. In one embodiment, the PEG-lipid is PEG_(2k)-DMG.

In one embodiment, the lipid nanoparticles described herein can comprisea PEG lipid which is a non-diffusible PEG. Non-limiting examples ofnon-diffusible PEGs include PEG-DSG and PEG-DSPE.

PEG-lipids are known in the art, such as those described in U.S. Pat.No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which areincorporated herein by reference in their entirety.

In general, some of the other lipid components (e.g., PEG lipids) ofvarious formulae, described herein may be synthesized as describedInternational Patent Application No. PCT/US2016/000129, filed Dec. 10,2016, entitled “Compositions and Methods for Delivery of TherapeuticAgents,” which is incorporated by reference in its entirety.

The lipid component of a lipid nanoparticle composition may include oneor more molecules comprising polyethylene glycol, such as PEG orPEG-modified lipids. Such species may be alternately referred to asPEGylated lipids. A PEG lipid is a lipid modified with polyethyleneglycol. A PEG lipid may be selected from the non-limiting groupincluding PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments the PEG-modified lipids are a modified form of PEGDMG. PEG-DMG has the following structure:

In one embodiment, PEG lipids useful in the present invention can bePEGylated lipids described in International Publication No.WO2012099755, the contents of which is herein incorporated by referencein its entirety. Any of these exemplary PEG lipids described herein maybe modified to comprise a hydroxyl group on the PEG chain. In certainembodiments, the PEG lipid is a PEG-OH lipid. As generally definedherein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylatedlipid”) is a PEGylated lipid having one or more hydroxyl (—OH) groups onthe lipid. In certain embodiments, the PEG-OH lipid includes one or morehydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH orhydroxy-PEGylated lipid comprises an —OH group at the terminus of thePEG chain. Each possibility represents a separate embodiment of thepresent invention.

In certain embodiments, a PEG lipid useful in the present invention is acompound of Formula (VII). Provided herein are compounds of Formula(VII):

or salts thereof, wherein:

-   -   R³ is —OR^(O);    -   R^(O) is hydrogen, optionally substituted alkyl, or an oxygen        protecting group;    -   r is an integer between 1 and 100, inclusive;    -   L¹ is optionally substituted C₁₋₁₀ alkylene, wherein at least        one methylene of the optionally substituted C₁₋₁₀ alkylene is        independently replaced with optionally substituted        carbocyclylene, optionally substituted heterocyclylene,        optionally substituted arylene, optionally substituted        heteroarylene, —O—, —N(R^(N))—, —S—, —C(O)—, —C(O)N(R^(N))—,        —NR^(N)C(O)—, —C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—,        —NR^(N)C(O)O—, or —NR^(N)C(O)N(R^(N))—;    -   D is a moiety obtained by click chemistry or a moiety cleavable        under physiological conditions;    -   m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;        A is of the formula:

-   -   each instance of L² is independently a bond or optionally        substituted C₁₋₆ alkylene, wherein one methylene unit of the        optionally substituted C₁₋₆ alkylene is optionally replaced with        —O—, —N(R^(N))—, —S—, —C(O)—, —C(O)N(R^(N))—, —NR^(N)C(O)—,        —C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—, or        —NR^(N)C(O)N(R^(N))—;    -   each instance of R² is independently optionally substituted        C₁₋₃₀ alkyl, optionally substituted C₁₋₃₀ alkenyl, or optionally        substituted C₁₋₃₀ alkynyl; optionally wherein one or more        methylene units of R² are independently replaced with optionally        substituted carbocyclylene, optionally substituted        heterocyclylene, optionally substituted arylene, optionally        substituted heteroarylene, —N(R^(N)), —O—, —S—, C(O)—,        —C(O)N(R^(N))—, —NR^(N)C(O)—, —NR^(N)C(O)N(R^(N))—, —C(O)O—,        —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—, —C(O)S—,        —SC(O)—, —C(═NR^(N))—, —C(═NR^(N))N(R^(N))—, —NR^(N)C(═NR^(N))—,        —NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—, —C(S)N(R^(N))—,        —NR^(N)C(S)—, —NR^(N)C(S)N(R^(N))—, —S(O)—, —OS(O)—, —S(O)O—,        —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—, —N(R^(N))S(O)—,        —S(O)N(R^(N))—, —N(R^(N))S(O)N(R^(N))—, —OS(O)N(R^(N))—,        —N(R^(N))S(O)O—, —S(O)₂—, —N(R^(N))S(O)₂—, —S(O)₂N(R^(N))—,        —N(R^(N))S(O)₂N(R^(N))—, —OS(O)₂N(R^(N))—, or —N(R^(N))S(O)₂O—;    -   each instance of R^(N) is independently hydrogen, optionally        substituted alkyl, or a nitrogen protecting group;    -   Ring B is optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl, or        optionally substituted heteroaryl; and    -   p is 1 or 2.

In certain embodiments, the compound of Formula (VII) is a PEG-OH lipid(i.e., R³ is —OR^(O), and R^(O) is hydrogen). In certain embodiments,the compound of Formula (VII) is of Formula (VII-OH):

or a salt thereof.

In certain embodiments, D is a moiety obtained by click chemistry (e.g.,triazole). In certain embodiments, the compound of Formula (VII) is ofFormula (VII-a-1) or (VII-a-2):

or a salt thereof.

In certain embodiments, the compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof, whereins is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In certain embodiments, the compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, D is a moiety cleavable under physiologicalconditions (e.g., ester, amide, carbonate, carbamate, urea). In certainembodiments, a compound of Formula (VII) is of Formula (VII-b-1) or(VII-b-2):

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of Formula(VII-b-1-OH) or (VII-b-2-OH):

or a salt thereof.

In certain embodiments, the compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or salts thereof.

In certain embodiments, a PEG lipid useful in the present invention is aPEGylated fatty acid. In certain embodiments, a PEG lipid useful in thepresent invention is a compound of Formula (VIII). Provided herein arecompounds of Formula (VIII):

or a salts thereof, wherein:

-   -   R³ is —OR^(O);    -   R^(O) is hydrogen, optionally substituted alkyl or an oxygen        protecting group;    -   r is an integer between 1 and 100, inclusive;    -   R⁵ is optionally substituted C₁₀₋₄₀ alkyl, optionally        substituted C₁₀₋₄₀ alkenyl, or optionally substituted C₁₀₋₄₀        alkynyl; and optionally one or more methylene groups of R⁵ are        replaced with optionally substituted carbocyclylene, optionally        substituted heterocyclylene, optionally substituted arylene,        optionally substituted heteroarylene, —N(R^(N)), —O—, —S—,        C(O)—, —C(O)N(R^(N))—, —NR^(N)C(O)—, —NR^(N)C(O)N(R^(N))—,        —C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—,        —C(O)S—, —SC(O)—, —C(═NR^(N))—, —C(═NR^(N))N(R^(N))—,        —NR^(N)C(═NR^(N))—, —NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—,        —C(S)N(R^(N))—, —NR^(N)C(S)—, —NR^(N)C(S)N(R^(N))—, —OS(O)—,        —S(O)O—, —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—,        —N(R^(N))S(O)—, —S(O)N(R^(N))—, —N(R^(N))S(O)N(R^(N))—,        —OS(O)N(R^(N))—, —N(R^(N))S(O)O—, —S(O)₂—, —N(R^(N))S(O)₂—,        —S(O)₂N(R^(N))—, —N(R^(N))S(O)₂N(R^(N))—, —OS(O)₂N(R^(N))—, or        —N(R^(N))S(O)₂O—; and    -   each instance of R^(N) is independently hydrogen, optionally        substituted alkyl, or a nitrogen protecting group.

In certain embodiments, the compound of Formula (VIII) is of Formula(VIII-OH):

or a salt thereof. In some embodiments, r is 45.

In certain embodiments, a compound of Formula (VIII) is of one of thefollowing formulae:

or a salt thereof. In some embodiments, r is 45.

In yet other embodiments the compound of Formula (VIII) is:

or a salt thereof.

In one embodiment, the compound of Formula (VIII) is

In one embodiment, the amount of PEG-lipid in the lipid composition of apharmaceutical composition disclosed herein ranges from about 0 mol % toabout 5 mol %, from about 1 mol % to about 5 mol %, from about 2 mol %to about 5 mol %, from about 0 mol % to about 4 mol %, from about 1 mol% to about 4 mol %, from about 2 mol % to about 4 mol %, from about 0mol % to about 3 mol %, from about 1 mol % to about 3 mol %, from about2 mol % to about 3 mol %, from about 0 mol % to about 2 mol %, or fromabout 1 mol % to about 2 mol %.

In one embodiment, the amount of PEG-lipid in the lipid compositiondisclosed herein is about 2 mol %. In one embodiment, the amount ofPEG-lipid in the lipid composition disclosed herein is about 1.5 mol %.

In one embodiment, the amount of PEG-lipid in the lipid compositiondisclosed herein is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol %.

In some aspects, the lipid composition of the pharmaceuticalcompositions disclosed herein does not comprise a PEG-lipid.

(v) Other Ionizable Amino Lipids

The lipid composition of the pharmaceutical composition disclosed hereincan comprise one or more ionizable amino lipids in addition to a lipidaccording to formula (I), (III), (IV), (V), or (VI).

Ionizable lipids can be selected from the non-limiting group consistingof 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),(13Z,165Z)—N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)). In addition to these, an ionizable amino lipid canalso be a lipid including a cyclic amine group.

Ionizable lipids can also be the compounds disclosed in InternationalPublication No. WO 2017/075531 A1, hereby incorporated by reference inits entirety. For example, the ionizable amino lipids include, but notlimited to:

and any combination thereof.

Ionizable lipids can also be the compounds disclosed in InternationalPublication No. WO 2015/199952 A1, hereby incorporated by reference inits entirety. For example, the ionizable amino lipids include, but notlimited to:

and any combination thereof.

Ionizable lipids can further include, but are not limited to:

and any combination thereof.

(vi) Other Lipid Composition Components

The lipid composition of a pharmaceutical composition disclosed hereincan include one or more components in addition to those described above.For example, the lipid composition can include one or more permeabilityenhancer molecules, carbohydrates, polymers, surface altering agents(e.g., surfactants), or other components. For example, a permeabilityenhancer molecule can be a molecule described by U.S. Patent ApplicationPublication No. 2005/0222064. Carbohydrates can include simple sugars(e.g., glucose) and polysaccharides (e.g., glycogen and derivatives andanalogs thereof).

A polymer can be included in and/or used to encapsulate or partiallyencapsulate a pharmaceutical composition disclosed herein (e.g., apharmaceutical composition in lipid nanoparticle form). A polymer can bebiodegradable and/or biocompatible. A polymer can be selected from, butis not limited to, polyamines, polyethers, polyamides, polyesters,polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides,polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates.

The ratio between the lipid composition and the polynucleotide range canbe from about 10:1 to about 60:1 (wt/wt).

In some embodiments, the ratio between the lipid composition and thepolynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1,29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In someembodiments, the wt/wt ratio of the lipid composition to thepolynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

In some embodiments, the pharmaceutical composition disclosed herein cancontain more than one polypeptides. For example, a pharmaceuticalcomposition disclosed herein can contain two or more polynucleotides(e.g., RNA, e.g., mRNA).

In one embodiment, the lipid nanoparticles described herein can comprisepolynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or70:1, or a range or any of these ratios such as, but not limited to, 5:1to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, fromabout 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 toabout 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1,from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 toabout 25:1, from about 10:1 to about 30:1, from about 10:1 to about35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, fromabout 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about20:1, from about 15:1 to about 25:1, from about 15:1 to about 30:1, fromabout 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

In one embodiment, the lipid nanoparticles described herein can comprisethe polynucleotide in a concentration from approximately 0.1 mg/ml to 2mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml,1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

(vii) Nanoparticle Compositions

In some embodiments, the pharmaceutical compositions disclosed hereinare formulated as lipid nanoparticles (LNP). Accordingly, the presentdisclosure also provides nanoparticle compositions comprising (i) alipid composition comprising a delivery agent such as a compound offormula (I) or (III) as described herein, and (ii) a polynucleotideencoding a Citrin polypeptide. In such nanoparticle composition, thelipid composition disclosed herein can encapsulate the polynucleotideencoding a Citrin polypeptide.

Nanoparticle compositions are typically sized on the order ofmicrometers or smaller and can include a lipid bilayer. Nanoparticlecompositions encompass lipid nanoparticles (LNPs), liposomes (e.g.,lipid vesicles), and lipoplexes. For example, a nanoparticle compositioncan be a liposome having a lipid bilayer with a diameter of 500 nm orless.

Nanoparticle compositions include, for example, lipid nanoparticles(LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticlecompositions are vesicles including one or more lipid bilayers. Incertain embodiments, a nanoparticle composition includes two or moreconcentric bilayers separated by aqueous compartments. Lipid bilayerscan be functionalized and/or crosslinked to one another. Lipid bilayerscan include one or more ligands, proteins, or channels.

In some embodiments, the nanoparticle compositions of the presentdisclosure comprise at least one compound according to formula (I),(III), (IV), (V), or (VI). For example, the nanoparticle composition caninclude one or more of Compounds 1-147, or one or more of Compounds1-342. Nanoparticle compositions can also include a variety of othercomponents. For example, the nanoparticle composition can include one ormore other lipids in addition to a lipid according to formula (I),(III), (IV), (V), or (VI), such as (i) at least one phospholipid, (ii)at least one structural lipid, (iii) at least one PEG-lipid, or (iv) anycombination thereof. Inclusion of structural lipid can be optional, forexample when lipids according to formula III are used in the lipidnanoparticle compositions of the invention.

In some embodiments, the nanoparticle composition comprises a compoundof formula (I) (e.g., Compounds 18, 25, 26 or 48). In some embodiments,the nanoparticle composition comprises a compound of formula (I) (e.g.,Compounds 18, 25, 26 or 48) and a phospholipid (e.g., DSPC)

In some embodiments, the nanoparticle composition comprises a compoundof formula (III) (e.g., Compound 236). In some embodiments, thenanoparticle composition comprises a compound of formula (III) (e.g.,Compound 236) and a phospholipid (e.g., DOPE or DSPC).

In some embodiments, the nanoparticle composition comprises a lipidcomposition consisting or consisting essentially of compound of formula(I) (e.g., Compounds 18, 25, 26 or 48). In some embodiments, thenanoparticle composition comprises a lipid composition consisting orconsisting essentially of a compound of formula (I) (e.g., Compounds 18,25, 26 or 48) and a phospholipid (e.g., DSPC).

In some embodiments, the nanoparticle composition comprises a lipidcomposition consisting or consisting essentially of compound of formula(III) (e.g., Compound 236). In some embodiments, the nanoparticlecomposition comprises a lipid composition consisting or consistingessentially of a compound of formula (III) (e.g., Compound 236) and aphospholipid (e.g., DOPE or DSPC).

In one embodiment, a lipid nanoparticle comprises an ionizable lipid, astructural lipid, a phospholipid, and mRNA. In some embodiments, the LNPcomprises an ionizable lipid, a PEG-modified lipid, a sterol and astructural lipid. In some embodiments, the LNP has a molar ratio ofabout 20-60% ionizable lipid: about 5-25% structural lipid: about 25-55%sterol; and about 0.5-15% PEG-modified lipid. In some embodiments, theLNP comprises a molar ratio of about 50% ionizable lipid, about 1.5%PEG-modified lipid, about 38.5% cholesterol and about 10% structurallipid. In some embodiments, the LNP comprises a molar ratio of about 55%ionizable lipid, about 2.5% PEG lipid, about 32.5% cholesterol and about10% structural lipid. In some embodiments, the ionizable lipid is anionizable amino lipid and the structural lipid is a neutral lipid, andthe sterol is a cholesterol. In some embodiments, the LNP has a molarratio of 50:38.5:10:1.5 of ionizable lipid:cholesterol:DSPC:PEG lipid.In some embodiments, the ionizable lipid is Compound 18 or Compound 236,and the PEG lipid is Compound 428.

In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 ofCompound 18:Cholesterol:Phospholipid:Compound 428. In some embodiments,the LNP has a molar ratio of 50:38.5:10:1.5 of Compound18:Cholesterol:DSPC:Compound 428.

In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 ofCompound 236:Cholesterol:Phospholipid:Compound 428. In some embodiments,the LNP has a molar ratio of 50:38.5:10:1.5 of Compound236:Cholesterol:DSPC:Compound 428.

In some embodiments, the LNP has a polydispersity value of less than0.4. In some embodiments, the LNP has a net neutral charge at a neutralpH. In some embodiments, the LNP has a mean diameter of 50-150 nm. Insome embodiments, the LNP has a mean diameter of 80-100 nm.

As generally defined herein, the term “lipid” refers to a small moleculethat has hydrophobic or amphiphilic properties. Lipids may be naturallyoccurring or synthetic. Examples of classes of lipids include, but arenot limited to, fats, waxes, sterol-containing metabolites, vitamins,fatty acids, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids, and polyketides, and prenol lipids. In some instances,the amphiphilic properties of some lipids leads them to form liposomes,vesicles, or membranes in aqueous media.

In some embodiments, a lipid nanoparticle (LNP) may comprise anionizable lipid. As used herein, the term “ionizable lipid” has itsordinary meaning in the art and may refer to a lipid comprising one ormore charged moieties. In some embodiments, an ionizable lipid may bepositively charged or negatively charged. An ionizable lipid may bepositively charged, in which case it can be referred to as “cationiclipid”. In certain embodiments, an ionizable lipid molecule may comprisean amine group, and can be referred to as an ionizable amino lipid. Asused herein, a “charged moiety” is a chemical moiety that carries aformal electronic charge, e.g., monovalent (+1, or −1), divalent (+2, or−2), trivalent (+3, or −3), etc. The charged moiety may be anionic(i.e., negatively charged) or cationic (i.e., positively charged).Examples of positively-charged moieties include amine groups (e.g.,primary, secondary, and/or tertiary amines), ammonium groups, pyridiniumgroup, guanidine groups, and imidazolium groups. In a particularembodiment, the charged moieties comprise amine groups. Examples ofnegatively-charged groups or precursors thereof, include carboxylategroups, sulfonate groups, sulfate groups, phosphonate groups, phosphategroups, hydroxyl groups, and the like. The charge of the charged moietymay vary, in some cases, with the environmental conditions, for example,changes in pH may alter the charge of the moiety, and/or cause themoiety to become charged or uncharged. In general, the charge density ofthe molecule may be selected as desired.

It should be understood that the terms “charged” or “charged moiety”does not refer to a “partial negative charge” or “partial positivecharge” on a molecule. The terms “partial negative charge” and “partialpositive charge” are given its ordinary meaning in the art. A “partialnegative charge” may result when a functional group comprises a bondthat becomes polarized such that electron density is pulled toward oneatom of the bond, creating a partial negative charge on the atom. Thoseof ordinary skill in the art will, in general, recognize bonds that canbecome polarized in this way.

In some embodiments, the ionizable lipid is an ionizable amino lipid,sometimes referred to in the art as an “ionizable cationic lipid”. Inone embodiment, the ionizable amino lipid may have a positively chargedhydrophilic head and a hydrophobic tail that are connected via a linkerstructure.

In addition to these, an ionizable lipid may also be a lipid including acyclic amine group.

In one embodiment, the ionizable lipid may be selected from, but notlimited to, a ionizable lipid described in International PublicationNos. WO2013086354 and WO2013116126; the contents of each of which areherein incorporated by reference in their entirety.

In yet another embodiment, the ionizable lipid may be selected from, butnot limited to, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969; each ofwhich is herein incorporated by reference in their entirety.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, hereinincorporated by reference in its entirety. In one embodiment, the lipidmay be synthesized by methods known in the art and/or as described inInternational Publication Nos. WO2013086354; the contents of each ofwhich are herein incorporated by reference in their entirety.

Nanoparticle compositions can be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) can be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) can beused to measure zeta potentials. Dynamic light scattering can also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential.

The size of the nanoparticles can help counter biological reactions suchas, but not limited to, inflammation, or can increase the biologicaleffect of the polynucleotide.

As used herein, “size” or “mean size” in the context of nanoparticlecompositions refers to the mean diameter of a nanoparticle composition.

In one embodiment, the polynucleotide encoding a Citrin polypeptide areformulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In one embodiment, the nanoparticles have a diameter from about 10 to500 nm. In one embodiment, the nanoparticle has a diameter greater than100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm,greater than 300 nm, greater than 350 nm, greater than 400 nm, greaterthan 450 nm, greater than 500 nm, greater than 550 nm, greater than 600nm, greater than 650 nm, greater than 700 nm, greater than 750 nm,greater than 800 nm, greater than 850 nm, greater than 900 nm, greaterthan 950 nm or greater than 1000 nm.

In some embodiments, the largest dimension of a nanoparticle compositionis 1 μm or shorter (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, orshorter).

A nanoparticle composition can be relatively homogenous. Apolydispersity index can be used to indicate the homogeneity of ananoparticle composition, e.g., the particle size distribution of thenanoparticle composition. A small (e.g., less than 0.3) polydispersityindex generally indicates a narrow particle size distribution. Ananoparticle composition can have a polydispersity index from about 0 toabout 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersityindex of a nanoparticle composition disclosed herein can be from about0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential can describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species caninteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition disclosed herein can be from about −10 mV to about +20 mV,from about −10 mV to about +15 mV, from about 10 mV to about +10 mV,from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, fromabout −10 mV to about −5 mV, from about −5 mV to about +20 mV, fromabout −5 mV to about +15 mV, from about −5 mV to about +10 mV, fromabout −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV toabout +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about+20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about+10 mV.

In some embodiments, the zeta potential of the lipid nanoparticles canbe from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, fromabout 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV toabout 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV,from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, fromabout 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mVto about 30 mV, from about 10 mV to about 20 mV, from about 20 mV toabout 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV,from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, fromabout 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mVto about 70 mV, from about 30 mV to about 60 mV, from about 30 mV toabout 50 mV, from about 30 mV to about 40 mV, from about 40 mV to about100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV,and from about 40 mV to about 50 mV. In some embodiments, the zetapotential of the lipid nanoparticles can be from about 10 mV to about 50mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV,and from about 25 mV to about 35 mV. In some embodiments, the zetapotential of the lipid nanoparticles can be about 10 mV, about 20 mV,about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about80 mV, about 90 mV, and about 100 mV.

The term “encapsulation efficiency” of a polynucleotide describes theamount of the polynucleotide that is encapsulated by or otherwiseassociated with a nanoparticle composition after preparation, relativeto the initial amount provided. As used herein, “encapsulation” canrefer to complete, substantial, or partial enclosure, confinement,surrounding, or encasement.

Encapsulation efficiency is desirably high (e.g., close to 100%). Theencapsulation efficiency can be measured, for example, by comparing theamount of the polynucleotide in a solution containing the nanoparticlecomposition before and after breaking up the nanoparticle compositionwith one or more organic solvents or detergents.

Fluorescence can be used to measure the amount of free polynucleotide ina solution. For the nanoparticle compositions described herein, theencapsulation efficiency of a polynucleotide can be at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulationefficiency can be at least 80%. In certain embodiments, theencapsulation efficiency can be at least 90%.

The amount of a polynucleotide present in a pharmaceutical compositiondisclosed herein can depend on multiple factors such as the size of thepolynucleotide, desired target and/or application, or other propertiesof the nanoparticle composition as well as on the properties of thepolynucleotide.

For example, the amount of an mRNA useful in a nanoparticle compositioncan depend on the size (expressed as length, or molecular mass),sequence, and other characteristics of the mRNA. The relative amounts ofa polynucleotide in a nanoparticle composition can also vary.

The relative amounts of the lipid composition and the polynucleotidepresent in a lipid nanoparticle composition of the present disclosurecan be optimized according to considerations of efficacy andtolerability. For compositions including an mRNA as a polynucleotide,the N:P ratio can serve as a useful metric.

As the N:P ratio of a nanoparticle composition controls both expressionand tolerability, nanoparticle compositions with low N:P ratios andstrong expression are desirable. N:P ratios vary according to the ratioof lipids to RNA in a nanoparticle composition.

In general, a lower N:P ratio is preferred. The one or more RNA, lipids,and amounts thereof can be selected to provide an N:P ratio from about2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. Incertain embodiments, the N:P ratio can be from about 2:1 to about 8:1.In other embodiments, the N:P ratio is from about 5:1 to about 8:1. Incertain embodiments, the N:P ratio is between 5:1 and 6:1. In onespecific aspect, the N:P ratio is about is about 5.67:1.

In addition to providing nanoparticle compositions, the presentdisclosure also provides methods of producing lipid nanoparticlescomprising encapsulating a polynucleotide. Such method comprises usingany of the pharmaceutical compositions disclosed herein and producinglipid nanoparticles in accordance with methods of production of lipidnanoparticles known in the art. See, e.g., Wang et al. (2015) “Deliveryof oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev.87:68-80; Silva et al. (2015) “Delivery Systems for Biopharmaceuticals.Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol. 16:940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles andNanostructured Lipid Carriers: Structure, Preparation and Application”Adv. Pharm. Bull. 5:305-13; Silva et al. (2015) “Lipid nanoparticles forthe delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302,and references cited therein.

23. Other Delivery Agents

a. Liposomes, Lipoplexes, and Lipid Nanoparticles

In some embodiments, the compositions or formulations of the presentdisclosure comprise a delivery agent, e.g., a liposome, a lioplexes, alipid nanoparticle, or any combination thereof. The polynucleotidesdescribed herein (e.g., a polynucleotide comprising a nucleotidesequence encoding a Citrin polypeptide) can be formulated using one ormore liposomes, lipoplexes, or lipid nanoparticles. Liposomes,lipoplexes, or lipid nanoparticles can be used to improve the efficacyof the polynucleotides directed protein production as these formulationscan increase cell transfection by the polynucleotide; and/or increasethe translation of encoded protein. The liposomes, lipoplexes, or lipidnanoparticles can also be used to increase the stability of thepolynucleotides.

Liposomes are artificially-prepared vesicles that can primarily becomposed of a lipid bilayer and can be used as a delivery vehicle forthe administration of pharmaceutical formulations. Liposomes can be ofdifferent sizes. A multilamellar vesicle (MLV) can be hundreds ofnanometers in diameter, and can contain a series of concentric bilayersseparated by narrow aqueous compartments. A small unicellular vesicle(SUV) can be smaller than 50 nm in diameter, and a large unilamellarvesicle (LUV) can be between 50 and 500 nm in diameter. Liposome designcan include, but is not limited to, opsonins or ligands to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes can contain a low or ahigh pH value in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes can depend on the pharmaceutical formulationentrapped and the liposomal ingredients, the nature of the medium inwhich the lipid vesicles are dispersed, the effective concentration ofthe entrapped substance and its potential toxicity, any additionalprocesses involved during the application and/or delivery of thevesicles, the optimal size, polydispersity and the shelf-life of thevesicles for the intended application, and the batch-to-batchreproducibility and scale up production of safe and efficient liposomalproducts, etc.

As a non-limiting example, liposomes such as synthetic membrane vesiclescan be prepared by the methods, apparatus and devices described in U.S.Pub. Nos. US20130177638, US20130177637, US20130177636, US20130177635,US20130177634, US20130177633, US20130183375, US20130183373, andUS20130183372. In some embodiments, the polynucleotides described hereincan be encapsulated by the liposome and/or it can be contained in anaqueous core that can then be encapsulated by the liposome as describedin, e.g., Intl. Pub. Nos. WO2012031046, WO2012031043, WO2012030901,WO2012006378, and WO2013086526; and U.S. Pub. Nos. US20130189351,US20130195969 and US20130202684. Each of the references in hereinincorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a cationic oil-in-water emulsion where the emulsionparticle comprises an oil core and a cationic lipid that can interactwith the polynucleotide anchoring the molecule to the emulsion particle.In some embodiments, the polynucleotides described herein can beformulated in a water-in-oil emulsion comprising a continuoushydrophobic phase in which the hydrophilic phase is dispersed. Exemplaryemulsions can be made by the methods described in Intl. Pub. Nos.WO2012006380 and WO201087791, each of which is herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a lipid-polycation complex. The formation of thelipid-polycation complex can be accomplished by methods as described in,e.g., U.S. Pub. No. US20120178702. As a non-limiting example, thepolycation can include a cationic peptide or a polypeptide such as, butnot limited to, polylysine, polyornithine and/or polyarginine and thecationic peptides described in Intl. Pub. No. WO2012013326 or U.S. Pub.No. US20130142818. Each of the references is herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a lipid nanoparticle (LNP) such as those described inIntl. Pub. Nos. WO2013123523, WO2012170930, WO2011127255 andWO2008103276; and U.S. Pub. No. US20130171646, each of which is hereinincorporated by reference in its entirety.

Lipid nanoparticle formulations typically comprise one or more lipids.In some embodiments, the lipid is a cationic or an ionizable lipid. Insome embodiments, the lipid is an ionizable lipid (e.g., an ionizableamino lipid), sometimes referred to in the art as an “ionizable cationiclipid”. In some embodiments, lipid nanoparticle formulations furthercomprise other components, including a phospholipid, a structural lipidand a molecule capable of reducing particle aggregation, for example aPEG or PEG-modified lipid.

Exemplary ionizable lipids include, but not limited to, any one ofCompounds 1-342 disclosed herein, DLin-MC3-DMA (MC3), DLin-DMA, DLenDMA,DLin-D-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-K-DMA, DLin-KC2-DMA,DLin-KC3-DMA, DLin-KC4-DMA, DLin-C2K-DMA, DLin-MP-DMA, DODMA, 98N12-5,C12-200, DLin-C-DAP, DLin-DAC, DLinDAP, DLinAP, DLin-EG-DMA,DLin-2-DMAP, KL10, KL22, KL25, Octyl-CLinDMA, Octyl-CLinDMA (2R),Octyl-CLinDMA (2S), and any combination thereof. Other exemplaryionizable lipids include,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608),(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(16Z,19Z)—N5N-dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl] ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine,and (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine, and anycombination thereof.

Phospholipids include, but are not limited to, glycerophospholipids suchas phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.Phospholipids also include phosphosphingolipid, such as sphingomyelin.In some embodiments, the phospholipids are DLPC, DMPC, DOPC, DPPC, DSPC,DUPC, 18:0 Diether PC, DLnPC, DAPC, DHAPC, DOPE, 4ME 16:0 PE, DSPE,DLPE, DLnPE, DAPE, DHAPE, DOPG, and any combination thereof. In someembodiments, the phospholipids are MPPC, MSPC, PMPC, PSPC, SMPC, SPPC,DHAPE, DOPG, and any combination thereof. In some embodiments, theamount of phospholipids (e.g., DSPC) in the lipid composition rangesfrom about 1 mol % to about 20 mol %.

The structural lipids include sterols and lipids containing sterolmoieties. In some embodiments, the structural lipids includecholesterol, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid,alpha-tocopherol, and mixtures thereof. In some embodiments, thestructural lipid is cholesterol. In some embodiments, the amount of thestructural lipids (e.g., cholesterol) in the lipid composition rangesfrom about 20 mol % to about 60 mol %.

The PEG-modified lipids include PEG-modified phosphatidylethanolamineand phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 orPEG-CerC20), PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Such lipids are also referred to asPEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments,the PEG-lipid are 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol(PEG-DMG),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl,PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG),PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments,the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or20,000 daltons. In some embodiments, the amount of PEG-lipid in thelipid composition ranges from about 0 mol % to about 5 mol %.

In some embodiments, the LNP formulations described herein canadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in U.S. Pub. No.US20050222064, herein incorporated by reference in its entirety.

The LNP formulations can further contain a phosphate conjugate. Thephosphate conjugate can increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatescan be made by the methods described in, e.g., Intl. Pub. No.WO2013033438 or U.S. Pub. No. US20130196948. The LNP formulation canalso contain a polymer conjugate (e.g., a water soluble conjugate) asdescribed in, e.g., U.S. Pub. Nos. US20130059360, US20130196948, andUS20130072709. Each of the references is herein incorporated byreference in its entirety.

The LNP formulations can comprise a conjugate to enhance the delivery ofnanoparticles of the present invention in a subject. Further, theconjugate can inhibit phagocytic clearance of the nanoparticles in asubject. In some embodiments, the conjugate can be a “self” peptidedesigned from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al, Science 2013 339, 971-975,herein incorporated by reference in its entirety). As shown by Rodriguezet al. the self peptides delayed macrophage-mediated clearance ofnanoparticles which enhanced delivery of the nanoparticles.

The LNP formulations can comprise a carbohydrate carrier. As anon-limiting example, the carbohydrate carrier can include, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin (e.g., Intl. Pub. No.WO2012109121, herein incorporated by reference in its entirety).

The LNP formulations can be coated with a surfactant or polymer toimprove the delivery of the particle. In some embodiments, the LNP canbe coated with a hydrophilic coating such as, but not limited to, PEGcoatings and/or coatings that have a neutral surface charge as describedin U.S. Pub. No. US20130183244, herein incorporated by reference in itsentirety.

The LNP formulations can be engineered to alter the surface propertiesof particles so that the lipid nanoparticles can penetrate the mucosalbarrier as described in U.S. Pat. No. 8,241,670 or Intl. Pub. No.WO2013110028, each of which is herein incorporated by reference in itsentirety.

The LNP engineered to penetrate mucus can comprise a polymeric material(i.e., a polymeric core) and/or a polymer-vitamin conjugate and/or atri-block co-polymer. The polymeric material can include, but is notlimited to, polyamines, polyethers, polyamides, polyesters,polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides,polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates.

LNP engineered to penetrate mucus can also include surface alteringagents such as, but not limited to, polynucleotides, anionic proteins(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosindornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase.

In some embodiments, the mucus penetrating LNP can be a hypotonicformulation comprising a mucosal penetration enhancing coating. Theformulation can be hypotonic for the epithelium to which it is beingdelivered. Non-limiting examples of hypotonic formulations can be foundin, e.g., Intl. Pub. No. WO2013110028, herein incorporated by referencein its entirety.

In some embodiments, the polynucleotide described herein is formulatedas a lipoplex, such as, without limitation, the ATUPLEX™ system, theDACC system, the DBTC system and other siRNA-lipoplex technology fromSilence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT®(Cambridge, Mass.), and polyethylenimine (PEI) or protamine-basedtargeted and non-targeted delivery of nucleic acids (Aleku et al. CancerRes. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 201250:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al.,Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 201023:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293Weide et al. JImmunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188;Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095-4100;deFougerolles Hum Gene Ther. 2008 19:125-132; all of which areincorporated herein by reference in its entirety).

In some embodiments, the polynucleotides described herein are formulatedas a solid lipid nanoparticle (SLN), which can be spherical with anaverage diameter between 10 to 1000 nm. SLN possess a solid lipid corematrix that can solubilize lipophilic molecules and can be stabilizedwith surfactants and/or emulsifiers. Exemplary SLN can be those asdescribed in Intl. Pub. No. WO2013105101, herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated for controlled release and/or targeted delivery. As usedherein, “controlled release” refers to a pharmaceutical composition orcompound release profile that conforms to a particular pattern ofrelease to effect a therapeutic outcome. In one embodiment, thepolynucleotides can be encapsulated into a delivery agent describedherein and/or known in the art for controlled release and/or targeteddelivery. As used herein, the term “encapsulate” means to enclose,surround or encase. As it relates to the formulation of the compounds ofthe invention, encapsulation can be substantial, complete or partial.The term “substantially encapsulated” means that at least greater than50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of thepharmaceutical composition or compound of the invention can be enclosed,surrounded or encased within the delivery agent. “Partiallyencapsulation” means that less than 10, 10, 20, 30, 40 50 or less of thepharmaceutical composition or compound of the invention can be enclosed,surrounded or encased within the delivery agent.

Advantageously, encapsulation can be determined by measuring the escapeor the activity of the pharmaceutical composition or compound of theinvention using fluorescence and/or electron micrograph. For example, atleast 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,or greater than 99% of the pharmaceutical composition or compound of theinvention are encapsulated in the delivery agent.

In some embodiments, the polynucleotide controlled release formulationcan include at least one controlled release coating (e.g., OPADRY®,EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such asethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®)). In someembodiments, the polynucleotide controlled release formulation cancomprise a polymer system as described in U.S. Pub. No. US20130130348,or a PEG and/or PEG related polymer derivative as described in U.S. Pat.No. 8,404,222, each of which is incorporated by reference in itsentirety.

In some embodiments, the polynucleotides described herein can beencapsulated in a therapeutic nanoparticle, referred to herein as“therapeutic nanoparticle polynucleotides.” Therapeutic nanoparticlescan be formulated by methods described in, e.g., Intl. Pub. Nos.WO2010005740, WO2010030763, WO2010005721, WO2010005723, andWO2012054923; and U.S. Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286,US20120288541, US20120140790, US20130123351 and US20130230567; and U.S.Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211, each of whichis herein incorporated by reference in its entirety.

In some embodiments, the therapeutic nanoparticle polynucleotide can beformulated for sustained release. As used herein, “sustained release”refers to a pharmaceutical composition or compound that conforms to arelease rate over a specific period of time. The period of time caninclude, but is not limited to, hours, days, weeks, months and years. Asa non-limiting example, the sustained release nanoparticle of thepolynucleotides described herein can be formulated as disclosed in Intl.Pub. No. WO2010075072 and U.S. Pub. Nos. US20100216804, US20110217377,US20120201859 and US20130150295, each of which is herein incorporated byreference in their entirety.

In some embodiments, the therapeutic nanoparticle polynucleotide can beformulated to be target specific, such as those described in Intl. Pub.Nos. WO2008121949, WO2010005726, WO2010005725, WO2011084521 andWO2011084518; and U.S. Pub. Nos. US20100069426, US20120004293 andUS20100104655, each of which is herein incorporated by reference in itsentirety.

The LNPs can be prepared using microfluidic mixers or micromixers.Exemplary microfluidic mixers can include, but are not limited to, aslit interdigitial micromixer including, but not limited to thosemanufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or astaggered herringbone micromixer (SHM) (see Zhigaltsev et al.,“Bottom-up design and synthesis of limit size lipid nanoparticle systemswith aqueous and triglyceride cores using millisecond microfluidicmixing,” Langmuir 28:3633-40 (2012); Belliveau et al., “Microfluidicsynthesis of highly potent limit-size lipid nanoparticles for in vivodelivery of siRNA,” Molecular Therapy-Nucleic Acids. 1:e37 (2012); Chenet al., “Rapid discovery of potent siRNA-containing lipid nanoparticlesenabled by controlled microfluidic formulation,” J. Am. Chem. Soc.134(16):6948-51 (2012); each of which is herein incorporated byreference in its entirety). Exemplary micromixers include SlitInterdigital Microstructured Mixer (SIMM-V2) or a Standard SlitInterdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet(IJMM,) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany. Insome embodiments, methods of making LNP using SHM further comprisemixing at least two input streams wherein mixing occurs bymicrostructure-induced chaotic advection (MICA). According to thismethod, fluid streams flow through channels present in a herringbonepattern causing rotational flow and folding the fluids around eachother. This method can also comprise a surface for fluid mixing whereinthe surface changes orientations during fluid cycling. Methods ofgenerating LNPs using SHM include those disclosed in U.S. Pub. Nos.US20040262223 and US20120276209, each of which is incorporated herein byreference in their entirety.

In some embodiments, the polynucleotides described herein can beformulated in lipid nanoparticles using microfluidic technology (seeWhitesides, George M., “The Origins and the Future of Microfluidics,”Nature 442: 368-373 (2006); and Abraham et al., “Chaotic Mixer forMicrochannels,” Science 295: 647-651 (2002); each of which is hereinincorporated by reference in its entirety). In some embodiments, thepolynucleotides can be formulated in lipid nanoparticles using amicromixer chip such as, but not limited to, those from HarvardApparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). Amicromixer chip can be used for rapid mixing of two or more fluidstreams with a split and recombine mechanism.

In some embodiments, the polynucleotides described herein can beformulated in lipid nanoparticles having a diameter from about 1 nm toabout 100 nm such as, but not limited to, about 1 nm to about 20 nm,from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, fromabout 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm toabout 90 nm, from about 5 nm to about from 100 nm, from about 5 nm toabout 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm,from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, fromabout 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 20nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 toabout 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm,about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 toabout 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm,about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 toabout 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/orabout 90 to about 100 nm.

In some embodiments, the lipid nanoparticles can have a diameter fromabout 10 to 500 nm. In one embodiment, the lipid nanoparticle can have adiameter greater than 100 nm, greater than 150 nm, greater than 200 nm,greater than 250 nm, greater than 300 nm, greater than 350 nm, greaterthan 400 nm, greater than 450 nm, greater than 500 nm, greater than 550nm, greater than 600 nm, greater than 650 nm, greater than 700 nm,greater than 750 nm, greater than 800 nm, greater than 850 nm, greaterthan 900 nm, greater than 950 nm or greater than 1000 nm.

In some embodiments, the polynucleotides can be delivered using smallerLNPs. Such particles can comprise a diameter from below 0.1 μm up to 100nm such as, but not limited to, less than 0.1 μm, less than 1.0 μm, lessthan 5 μm, less than 10 μm, less than 15 um, less than 20 um, less than25 um, less than 30 um, less than 35 um, less than 40 um, less than 50um, less than 55 um, less than 60 um, less than 65 um, less than 70 um,less than 75 um, less than 80 um, less than 85 um, less than 90 um, lessthan 95 um, less than 100 um, less than 125 um, less than 150 um, lessthan 175 um, less than 200 um, less than 225 um, less than 250 um, lessthan 275 um, less than 300 um, less than 325 um, less than 350 um, lessthan 375 um, less than 400 um, less than 425 um, less than 450 um, lessthan 475 um, less than 500 um, less than 525 um, less than 550 um, lessthan 575 um, less than 600 um, less than 625 um, less than 650 um, lessthan 675 um, less than 700 um, less than 725 um, less than 750 um, lessthan 775 um, less than 800 um, less than 825 um, less than 850 um, lessthan 875 um, less than 900 um, less than 925 um, less than 950 um, orless than 975 um.

The nanoparticles and microparticles described herein can begeometrically engineered to modulate macrophage and/or the immuneresponse. The geometrically engineered particles can have varied shapes,sizes and/or surface charges to incorporate the polynucleotidesdescribed herein for targeted delivery such as, but not limited to,pulmonary delivery (see, e.g., Intl. Pub. No. WO2013082111, hereinincorporated by reference in its entirety). Other physical features thegeometrically engineering particles can include, but are not limited to,fenestrations, angled arms, asymmetry and surface roughness, charge thatcan alter the interactions with cells and tissues.

In some embodiment, the nanoparticles described herein are stealthnanoparticles or target-specific stealth nanoparticles such as, but notlimited to, those described in U.S. Pub. No. US20130172406, hereinincorporated by reference in its entirety. The stealth ortarget-specific stealth nanoparticles can comprise a polymeric matrix,which can comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates, or combinationsthereof.

b. Lipidoids

In some embodiments, the compositions or formulations of the presentdisclosure comprise a delivery agent, e.g., a lipidoid. Thepolynucleotides described herein (e.g., a polynucleotide comprising anucleotide sequence encoding a Citrin polypeptide) can be formulatedwith lipidoids. Complexes, micelles, liposomes or particles can beprepared containing these lipidoids and therefore to achieve aneffective delivery of the polynucleotide, as judged by the production ofan encoded protein, following the injection of a lipidoid formulationvia localized and/or systemic routes of administration. Lipidoidcomplexes of polynucleotides can be administered by various meansincluding, but not limited to, intravenous, intramuscular, orsubcutaneous routes.

The synthesis of lipidoids is described in literature (see Mahon et al.,Bioconjug. Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al.,Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc NatlAcad Sci USA. 2011 108:12996-3001; all of which are incorporated hereinin their entireties).

Formulations with the different lipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-SLAP; also known as 98N12-5, see Murugaiah et al., AnalyticalBiochemistry, 401:61 (2010)), C12-200 (including derivatives andvariants), and MD1, can be tested for in vivo activity. The lipidoid“98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879. Thelipidoid “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA.2010 107:1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670.Each of the references is herein incorporated by reference in itsentirety.

In one embodiment, the polynucleotides described herein can beformulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids can beprepared by the methods described in U.S. Pat. No. 8,450,298 (hereinincorporated by reference in its entirety).

The lipidoid formulations can include particles comprising either 3 or 4or more components in addition to polynucleotides. Lipidoids andpolynucleotide formulations comprising lipidoids are described in Intl.Pub. No. WO 2015051214 (herein incorporated by reference in itsentirety.

c. Hyaluronidase

In some embodiments, the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) and hyaluronidase for injection (e.g., intramuscular orsubcutaneous injection). Hyaluronidase catalyzes the hydrolysis ofhyaluronan, which is a constituent of the interstitial barrier.Hyaluronidase lowers the viscosity of hyaluronan, thereby increasestissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440).Alternatively, the hyaluronidase can be used to increase the number ofcells exposed to the polynucleotides administered intramuscularly orsubcutaneously.

d. Nanoparticle Mimics

In some embodiments, the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) is encapsulated within and/or absorbed to a nanoparticlemimic. A nanoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example, thepolynucleotides described herein can be encapsulated in a non-vironparticle that can mimic the delivery function of a virus (see e.g.,Intl. Pub. No. WO2012006376 and U.S. Pub. Nos. US20130171241 andUS20130195968, each of which is herein incorporated by reference in itsentirety).

e. Nanotubes

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) attached or otherwise bound to (e.g., through steric,ionic, covalent and/or other forces) at least one nanotube, such as, butnot limited to, rosette nanotubes, rosette nanotubes having twin baseswith a linker, carbon nanotubes and/or single-walled carbon nanotubes.Nanotubes and nanotube formulations comprising a polynucleotide aredescribed in, e.g., Intl. Pub. No. WO2014152211, herein incorporated byreference in its entirety.

f. Self-Assembled Nanoparticles, or Self-Assembled Macromolecules

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in self-assembled nanoparticles, or amphiphilicmacromolecules (AMs) for delivery. AMs comprise biocompatibleamphiphilic polymers that have an alkylated sugar backbone covalentlylinked to poly(ethylene glycol). In aqueous solution, the AMsself-assemble to form micelles. Nucleic acid self-assemblednanoparticles are described in Intl. Appl. No. PCT/US2014/027077, andAMs and methods of forming AMs are described in U.S. Pub. No.US20130217753, each of which is herein incorporated by reference in itsentirety.

g. Inorganic Nanoparticles, Semi-Conductive and Metallic Nanoparticles

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in inorganic nanoparticles, or water-dispersiblenanoparticles comprising a semiconductive or metallic material. Theinorganic nanoparticles can include, but are not limited to, claysubstances that are water swellable. The water-dispersible nanoparticlescan be hydrophobic or hydrophilic nanoparticles. As a non-limitingexample, the inorganic, semi-conductive and metallic nanoparticles aredescribed in, e.g., U.S. Pat. Nos. 5,585,108 and 8,257,745; and U.S.Pub. Nos. US20120228565, US 20120265001 and US 20120283503, each ofwhich is herein incorporated by reference in their entirety.

h. Surgical Sealants: Gels and Hydrogels

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in a surgical sealant. Surgical sealants such as gels andhydrogels are described in Intl. Appl. No. PCT/US2014/027077, hereinincorporated by reference in its entirety.

i. Suspension Formulations

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in suspensions. In some embodiments, suspensions comprise apolynucleotide, water immiscible oil depots, surfactants and/orco-surfactants and/or co-solvents. Suspensions can be formed by firstpreparing an aqueous solution of a polynucleotide and an oil-based phasecomprising one or more surfactants, and then mixing the two phases(aqueous and oil-based).

Exemplary oils for suspension formulations can include, but are notlimited to, sesame oil and Miglyol (comprising esters of saturatedcoconut and palm kernel oil-derived caprylic and capric fatty acids andglycerin or propylene glycol), corn oil, soybean oil, peanut oil,beeswax and/or palm seed oil. Exemplary surfactants can include, but arenot limited to Cremophor, polysorbate 20, polysorbate 80, polyethyleneglycol, transcutol, Capmul®, labrasol, isopropyl myristate, and/or Span80. In some embodiments, suspensions can comprise co-solvents including,but not limited to ethanol, glycerol and/or propylene glycol.

In some embodiments, suspensions can provide modulation of the releaseof the polynucleotides into the surrounding environment by diffusionfrom a water immiscible depot followed by resolubilization into asurrounding environment (e.g., an aqueous environment).

In some embodiments, the polynucleotides can be formulated such thatupon injection, an emulsion forms spontaneously (e.g., when delivered toan aqueous phase), which can provide a high surface area to volume ratiofor release of polynucleotides from an oil phase to an aqueous phase. Insome embodiments, the polynucleotide is formulated in a nanoemulsion,which can comprise a liquid hydrophobic core surrounded by or coatedwith a lipid or surfactant layer. Exemplary nanoemulsions and theirpreparations are described in, e.g., U.S. Pat. No. 8,496,945, hereinincorporated by reference in its entirety.

j. Cations and Anions

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) and a cation or anion, such as Zn2+, Ca2+, Cu2+, Mg2+ andcombinations thereof. Exemplary formulations can include polymers and apolynucleotide complexed with a metal cation as described in, e.g., U.S.Pat. Nos. 6,265,389 and 6,555,525, each of which is herein incorporatedby reference in its entirety. In some embodiments, cationicnanoparticles can contain a combination of divalent and monovalentcations. The delivery of polynucleotides in cationic nanoparticles or inone or more depot comprising cationic nanoparticles can improvepolynucleotide bioavailability by acting as a long-acting depot and/orreducing the rate of degradation by nucleases.

k. Molded Nanoparticles and Microparticles

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in molded nanoparticles in various sizes, shapes andchemistry. For example, the nanoparticles and/or microparticles can bemade using the PRINT® technology by LIQUIDA TECHNOLOGIES® (Morrisville,N.C.) (e.g., International Pub. No. WO2007024323, herein incorporated byreference in its entirety).

In some embodiments, the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) is formulated in microparticles. The microparticles cancontain a core of the polynucleotide and a cortex of a biocompatibleand/or biodegradable polymer, including but not limited to,poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, apolyorthoester and a polyanhydride. The microparticle can have adsorbentsurfaces to adsorb polynucleotides. The microparticles can have adiameter of from at least 1 micron to at least 100 microns (e.g., atleast 1 micron, at least 10 micron, at least 20 micron, at least 30micron, at least 50 micron, at least 75 micron, at least 95 micron, andat least 100 micron). In some embodiment, the compositions orformulations of the present disclosure are microemulsions comprisingmicroparticles and polynucleotides. Exemplary microparticles,microemulsions and their preparations are described in, e.g., U.S. Pat.Nos. 8,460,709, 8,309,139 and 8,206,749; U.S. Pub. Nos. US20130129830,US2013195923 and US20130195898; and Intl. Pub. No. WO2013075068, each ofwhich is herein incorporated by reference in its entirety.

l. NanoJackets and NanoLiposomes

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in NanoJackets and NanoLiposomes by Keystone Nano (StateCollege, Pa.). NanoJackets are made of materials that are naturallyfound in the body including calcium, phosphate and can also include asmall amount of silicates. Nanojackets can have a size ranging from 5 to50 nm.

NanoLiposomes are made of lipids such as, but not limited to, lipidsthat naturally occur in the body. NanoLiposomes can have a size rangingfrom 60-80 nm. In some embodiments, the polynucleotides disclosed hereinare formulated in a NanoLiposome such as, but not limited to, CeramideNanoLiposomes.

m. Cells or Minicells

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) that is transfected ex vivo into cells, which aresubsequently transplanted into a subject. Cell-based formulations of thepolynucleotide disclosed herein can be used to ensure cell transfection(e.g., in the cellular carrier), alter the biodistribution of thepolynucleotide (e.g., by targeting the cell carrier to specific tissuesor cell types), and/or increase the translation of encoded protein.

Exemplary cells include, but are not limited to, red blood cells,virosomes, and electroporated cells (see e.g., Godfrin et al., ExpertOpin Biol Ther. 2012 12:127-133; Fang et al., Expert Opin Biol Ther.2012 12:385-389; Hu et al., Proc Natl Acad Sci USA. 2011108:10980-10985; Lund et al., Pharm Res. 2010 27:400-420; Huckriede etal., J Liposome Res. 2007; 17:39-47; Cusi, Hum Vaccin. 2006 2:1-7; deJonge et al., Gene Ther. 2006 13:400-411; all of which are hereinincorporated by reference in its entirety).

A variety of methods are known in the art and are suitable forintroduction of nucleic acid into a cell, including viral and non-viralmediated techniques. Examples of typical non-viral mediated techniquesinclude, but are not limited to, electroporation, calcium phosphatemediated transfer, nucleofection, sonoporation, heat shock,magnetofection, liposome mediated transfer, microinjection,microprojectile mediated transfer (nanoparticles), cationic polymermediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol(PEG) and the like) or cell fusion.

In some embodiments, the polynucleotides described herein can bedelivered in synthetic virus-like particles (VLPs) synthesized by themethods as described in Intl. Pub Nos. WO2011085231 and WO2013116656;and U.S. Pub. No. 20110171248, each of which is herein incorporated byreference in its entirety.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to delivernucleic acids in vivo (Yoon and Park, Expert Opin Drug Deliv. 20107:321-330; Postema and Gilja, Curr Pharm Biotechnol. 2007 8:355-361;Newman and Bettinger, Gene Ther. 2007 14:465-475; U.S. Pub. Nos.US20100196983 and US20100009424; all herein incorporated by reference intheir entirety).

In some embodiments, the polynucleotides described herein can bedelivered by electroporation. Electroporation techniques are known todeliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). Electroporation devicesare sold by many companies worldwide including, but not limited to BTX®Instruments (Holliston, Mass.) (e.g., the AgilePulse In Vivo System) andInovio (Blue Bell, Pa.) (e.g., Inovio SP-5P intramuscular deliverydevice or the CELLECTRA® 3000 intradermal delivery device).

In some embodiments, the cells are selected from the group consisting ofmammalian cells, bacterial cells, plant, microbial, algal and fungalcells. In some embodiments, the cells are mammalian cells, such as, butnot limited to, human, mouse, rat, goat, horse, rabbit, hamster or cowcells. In a further embodiment, the cells can be from an establishedcell line, including, but not limited to, HeLa, NS0, SP2/0, KEK 293T,Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jurkat, CHO-K1, DG44,CHOK1SV, CHO-S, Huvec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2,IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO)cells.

In certain embodiments, the cells are fungal cells, such as, but notlimited to, Chrysosporium cells, Aspergillus cells, Trichoderma cells,Dictyostelium cells, Candida cells, Saccharomyces cells,Schizosaccharomyces cells, and Penicillium cells.

In certain embodiments, the cells are bacterial cells such as, but notlimited to, E. coli, B. subtilis, or BL21 cells. Primary and secondarycells to be transfected by the methods of the invention can be obtainedfrom a variety of tissues and include, but are not limited to, all celltypes that can be maintained in culture. The primary and secondary cellsinclude, but are not limited to, fibroblasts, keratinocytes, epithelialcells (e.g., mammary epithelial cells, intestinal epithelial cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells andprecursors of these somatic cell types. Primary cells can also beobtained from a donor of the same species or from another species (e.g.,mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein in bacterialminicells. As a non-limiting example, bacterial minicells can be thosedescribed in Intl. Pub. No. WO2013088250 or U.S. Pub. No. US20130177499,each of which is herein incorporated by reference in its entirety.

n. Semi-Solid Compositions

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in a hydrophobic matrix to form a semi-solid or paste-likecomposition. As a non-limiting example, the semi-solid or paste-likecomposition can be made by the methods described in Intl. Pub. No.WO201307604, herein incorporated by reference in its entirety.

o. Exosomes

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in exosomes, which can be loaded with at least onepolynucleotide and delivered to cells, tissues and/or organisms. As anon-limiting example, the polynucleotides can be loaded in the exosomesas described in Intl. Pub. No. WO2013084000, herein incorporated byreference in its entirety.

p. Silk-Based Delivery

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) that is formulated for silk-based delivery. The silk-baseddelivery system can be formed by contacting a silk fibroin solution witha polynucleotide described herein. As a non-limiting example, asustained release silk-based delivery system and methods of making suchsystem are described in U.S. Pub. No. US20130177611, herein incorporatedby reference in its entirety.

q. Amino Acid Lipids

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) that is formulation with an amino acid lipid. Amino acidlipids are lipophilic compounds comprising an amino acid residue and oneor more lipophilic tails. Non-limiting examples of amino acid lipids andmethods of making amino acid lipids are described in U.S. Pat. No.8,501,824. The amino acid lipid formulations can deliver apolynucleotide in releasable form that comprises an amino acid lipidthat binds and releases the polynucleotides. As a non-limiting example,the release of the polynucleotides described herein can be provided byan acid-labile linker as described in, e.g., U.S. Pat. Nos. 7,098,032,6,897,196, 6,426,086, 7,138,382, 5,563,250, and 5,505,931, each of whichis herein incorporated by reference in its entirety.

r. Microvesicles

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in a microvesicle formulation. Exemplary microvesiclesinclude those described in U.S. Pub. No. US20130209544 (hereinincorporated by reference in its entirety). In some embodiments, themicrovesicle is an ARRDC1-mediated microvesicles (ARMMs) as described inIntl. Pub. No. WO2013119602 (herein incorporated by reference in itsentirety).

s. Interpolyelectrolyte Complexes

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in an interpolyelectrolyte complex. Interpolyelectrolytecomplexes are formed when charge-dynamic polymers are complexed with oneor more anionic molecules. Non-limiting examples of charge-dynamicpolymers and interpolyelectrolyte complexes and methods of makinginterpolyelectrolyte complexes are described in U.S. Pat. No. 8,524,368,herein incorporated by reference in its entirety.

t. Crystalline Polymeric Systems

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in crystalline polymeric systems. Crystalline polymericsystems are polymers with crystalline moieties and/or terminal unitscomprising crystalline moieties. Exemplary polymers are described inU.S. Pat. No. 8,524,259 (herein incorporated by reference in itsentirety).

u. Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) and a natural and/or synthetic polymer. The polymersinclude, but not limited to, polyethenes, polyethylene glycol (PEG),poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, elastic biodegradable polymer, biodegradablecopolymer, biodegradable polyester copolymer, biodegradable polyestercopolymer, multiblock copolymers, poly[α-(4-aminobutyl)-L-glycolic acid)(PAGA), biodegradable cross-linked cationic multi-block copolymers,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyureas, polystyrenes, polyamines,polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),amine-containing polymers, dextran polymers, dextran polymer derivativesor combinations thereof.

Exemplary polymers include, DYNAMIC POLYCONJUGATE® (Arrowhead ResearchCorp., Pasadena, Calif.) formulations from MIRUS® Bio (Madison, Wis.)and Roche Madison (Madison, Wis.), PHASERX™ polymer formulations suchas, without limitation, SMARTT POLYMER TECHNOLOGY™ (PHASERX®, Seattle,Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego,Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena,Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.) and pH responsive co-blockpolymers such as PHASERX® (Seattle, Wash.).

The polymer formulations allow a sustained or delayed release of thepolynucleotide (e.g., following intramuscular or subcutaneousinjection). The altered release profile for the polynucleotide canresult in, for example, translation of an encoded protein over anextended period of time. The polymer formulation can also be used toincrease the stability of the polynucleotide. Sustained releaseformulations can include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc. Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc. Deerfield, Ill.).

As a non-limiting example modified mRNA can be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradable,biocompatible polymers that are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C.

As a non-limiting example, the polynucleotides described herein can beformulated with the polymeric compound of PEG grafted with PLL asdescribed in U.S. Pat. No. 6,177,274. As another non-limiting example,the polynucleotides described herein can be formulated with a blockcopolymer such as a PLGA-PEG block copolymer (see e.g., U.S. Pub. No.US20120004293 and U.S. Pat. Nos. 8,236,330 and 8,246,968), or aPLGA-PEG-PLGA block copolymer (see e.g., U.S. Pat. No. 6,004,573). Eachof the references is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated with at least one amine-containing polymer such as, but notlimited to polylysine, polyethylene imine, poly(amidoamine) dendrimers,poly(amine-co-esters) or combinations thereof. Exemplary polyaminepolymers and their use as delivery agents are described in, e.g., U.S.Pat. Nos. 8,460,696, 8,236,280, each of which is herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a biodegradable cationic lipopolymer, a biodegradablepolymer, or a biodegradable copolymer, a biodegradable polyestercopolymer, a biodegradable polyester polymer, a linear biodegradablecopolymer, PAGA, a biodegradable cross-linked cationic multi-blockcopolymer or combinations thereof as described in, e.g., U.S. Pat. Nos.6,696,038, 6,517,869, 6,267,987, 6,217,912, 6,652,886, 8,057,821, and8,444,992; U.S. Pub. Nos. US20030073619, US20040142474, US20100004315,US2012009145 and US20130195920; and Intl Pub. Nos. WO2006063249 andWO2013086322, each of which is herein incorporated by reference in itsentirety.

In some embodiments, the polynucleotides described herein can beformulated in or with at least one cyclodextrin polymer as described inU.S. Pub. No. US20130184453. In some embodiments, the polynucleotidesdescribed herein can be formulated in or with at least one crosslinkedcation-binding polymers as described in Intl. Pub. Nos. WO2013106072,WO2013106073 and WO2013106086. In some embodiments, the polynucleotidesdescribed herein can be formulated in or with at least PEGylated albuminpolymer as described in U.S. Pub. No. US20130231287. Each of thereferences is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides disclosed herein can beformulated as a nanoparticle using a combination of polymers, lipids,and/or other biodegradable agents, such as, but not limited to, calciumphosphate. Components can be combined in a core-shell, hybrid, and/orlayer-by-layer architecture, to allow for fine-tuning of thenanoparticle for delivery (Wang et al., Nat Mater. 2006 5:791-796;Fuller et al., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv DrugDeliv Rev. 2011 63:748-761; Endres et al., Biomaterials. 201132:7721-7731; Su et al., Mol Pharm. 2011 Jun. 6; 8(3):774-87; hereinincorporated by reference in their entireties). As a non-limitingexample, the nanoparticle can comprise a plurality of polymers such as,but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA),hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (Intl. Pub.No. WO20120225129, herein incorporated by reference in its entirety).

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001; herein incorporated by reference in its entirety). Thecomplexation, delivery, and internalization of the polymericnanoparticles can be precisely controlled by altering the chemicalcomposition in both the core and shell components of the nanoparticle.For example, the core-shell nanoparticles can efficiently deliver siRNAto mouse hepatocytes after they covalently attach cholesterol to thenanoparticle.

In some embodiments, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG can be used to deliveryof the polynucleotides as described herein. In some embodiments, thelipid nanoparticles can comprise a core of the polynucleotides disclosedherein and a polymer shell, which is used to protect the polynucleotidesin the core. The polymer shell can be any of the polymers describedherein and are known in the art., the polymer shell can be used toprotect the polynucleotides in the core.

Core-shell nanoparticles for use with the polynucleotides describedherein are described in U.S. Pat. No. 8,313,777 or Intl. Pub. No.WO2013124867, each of which is herein incorporated by reference in theirentirety.

v. Peptides and Proteins

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) that is formulated with peptides and/or proteins toincrease transfection of cells by the polynucleotide, and/or to alterthe biodistribution of the polynucleotide (e.g., by targeting specifictissues or cell types), and/or increase the translation of encodedprotein (e.g., Intl. Pub. Nos. WO2012110636 and WO2013123298. In someembodiments, the peptides can be those described in U.S. Pub. Nos.US20130129726, US20130137644 and US20130164219. Each of the referencesis herein incorporated by reference in its entirety.

w. Conjugates

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) that is covalently linked to a carrier or targeting group,or including two encoding regions that together produce a fusion protein(e.g., bearing a targeting group and therapeutic protein or peptide) asa conjugate. The conjugate can be a peptide that selectively directs thenanoparticle to neurons in a tissue or organism, or assists in crossingthe blood-brain barrier.

The conjugates include a naturally occurring substance, such as aprotein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand can also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g., an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

In some embodiments, the conjugate can function as a carrier for thepolynucleotide disclosed herein. The conjugate can comprise a cationicpolymer such as, but not limited to, polyamine, polylysine,polyalkylenimine, and polyethylenimine that can be grafted to withpoly(ethylene glycol). Exemplary conjugates and their preparations aredescribed in U.S. Pat. No. 6,586,524 and U.S. Pub. No. US20130211249,each of which herein is incorporated by reference in its entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas an endothelial cell or bone cell. Targeting groups can also includehormones and hormone receptors. They can also include non-peptidicspecies, such as lipids, lectins, carbohydrates, vitamins, cofactors,multivalent lactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent frucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, or anactivator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein. As a non-limiting example, the targeting group can be aglutathione receptor (GR)-binding conjugate for targeted delivery acrossthe blood-central nervous system barrier as described in, e.g., U.S.Pub. No. US2013021661012 (herein incorporated by reference in itsentirety).

In some embodiments, the conjugate can be a synergisticbiomolecule-polymer conjugate, which comprises a long-actingcontinuous-release system to provide a greater therapeutic efficacy. Thesynergistic biomolecule-polymer conjugate can be those described in U.S.Pub. No. US20130195799. In some embodiments, the conjugate can be anaptamer conjugate as described in Intl. Pat. Pub. No. WO2012040524. Insome embodiments, the conjugate can be an amine containing polymerconjugate as described in U.S. Pat. No. 8,507,653. Each of thereferences is herein incorporated by reference in its entirety. In someembodiments, the polynucleotides can be conjugated to SMARTT POLYMERTECHNOLOGY® (PHASERX®, Inc. Seattle, Wash.).

In some embodiments, the polynucleotides described herein are covalentlyconjugated to a cell penetrating polypeptide, which can also include asignal sequence or a targeting sequence. The conjugates can be designedto have increased stability, and/or increased cell transfection; and/oraltered the biodistribution (e.g., targeted to specific tissues or celltypes).

In some embodiments, the polynucleotides described herein can beconjugated to an agent to enhance delivery. In some embodiments, theagent can be a monomer or polymer such as a targeting monomer or apolymer having targeting blocks as described in Intl. Pub. No.WO2011062965. In some embodiments, the agent can be a transport agentcovalently coupled to a polynucleotide as described in, e.g., U.S. Pat.Nos. 6,835.393 and 7,374,778. In some embodiments, the agent can be amembrane barrier transport enhancing agent such as those described inU.S. Pat. Nos. 7,737,108 and 8,003,129. Each of the references is hereinincorporated by reference in its entirety.

x. Micro-Organs

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in a micro-organ that can then express an encodedpolypeptide of interest in a long-lasting therapeutic formulation.Exemplary micro-organs and formulations are described in Intl. Pub. No.WO2014152211 (herein incorporated by reference in its entirety).

y. Pseudovirions

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding a Citrinpolypeptide) in pseudovirions (e.g., pseudovirions developed by AuraBiosciences, Cambridge, Mass.).

In some embodiments, the pseudovirion used for delivering thepolynucleotides can be derived from viruses such as, but not limited to,herpes and papillomaviruses as described in, e.g., U.S. Pub. Nos.US20130012450, US20130012566, US21030012426 and US20120207840; and Intl.Pub. No. WO2013009717, each of which is herein incorporated by referencein its entirety.

The pseudovirion can be a virus-like particle (VLP) prepared by themethods described in U.S. Pub. Nos. US20120015899 and US20130177587, andIntl. Pub. Nos. WO2010047839, WO2013116656, WO2013106525 andWO2013122262. In one aspect, the VLP can be bacteriophages MS, Q13, R17,fr, GA, Sp, MI, I, MXI, NL95, AP205, f2, PP7, and the plant virusesTurnip crinkle virus (TCV), Tomato bushy stunt virus (TBSV), Southernbean mosaic virus (SBMV) and members of the genus Bromovirus includingBroad bean mottle virus, Brome mosaic virus, Cassia yellow blotch virus,Cowpea chlorotic mottle virus (CCMV), Melandrium yellow fleck virus, andSpring beauty latent virus. In another aspect, the VLP can be derivedfrom the influenza virus as described in U.S. Pub. No. US20130177587 andU.S. Pat. No. 8,506,967. In one aspect, the VLP can comprise a B7-1and/or B7-2 molecule anchored to a lipid membrane or the exterior of theparticle such as described in Intl. Pub. No. WO2013116656. In oneaspect, the VLP can be derived from norovirus, rotavirus recombinant VP6protein or double layered VP2/VP6 such as the VLP as described in Intl.Pub. No. WO2012049366. Each of the references is herein incorporated byreference in its entirety.

In some embodiments, the pseudovirion can be a human papillomavirus-like particle as described in Intl. Pub. No. WO2010120266 and U.S.Pub. No. US20120171290. In some embodiments, the virus-like particle(VLP) can be a self-assembled particle. In one aspect, the pseudovirionscan be virion derived nanoparticles as described in U.S. Pub. Nos.US20130116408 and US20130115247; and Intl. Pub. No. WO2013119877. Eachof the references is herein incorporated by reference in their entirety.

Non-limiting examples of formulations and methods for formulating thepolynucleotides described herein are also provided in Intl. Pub. NoWO2013090648 (incorporated herein by reference in their entirety).

24. Accelerated Blood Clearance

The invention provides compounds, compositions and methods of usethereof for reducing the effect of ABC on a repeatedly administeredactive agent such as a biologically active agent. As will be readilyapparent, reducing or eliminating altogether the effect of ABC on anadministered active agent effectively increases its half-life and thusits efficacy.

In some embodiments the term reducing ABC refers to any reduction in ABCin comparison to a positive reference control ABC inducing LNP such asan MC3 LNP. ABC inducing LNPs cause a reduction in circulating levels ofan active agent upon a second or subsequent administration within agiven time frame. Thus a reduction in ABC refers to less clearance ofcirculating agent upon a second or subsequent dose of agent, relative toa standard LNP. The reduction may be, for instance, at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 100%. In some embodiments the reduction is 10-100%,10-50%, 20-100%, 20-50%, 30-100%, 30-50%, 40%-100%, 40-80%, 50-90%, or50-100%. Alternatively the reduction in ABC may be characterized as atleast a detectable level of circulating agent following a second orsubsequent administration or at least a 2 fold, 3 fold, 4 fold, 5 foldincrease in circulating agent relative to circulating agent followingadministration of a standard LNP. In some embodiments the reduction is a2-100 fold, 2-50 fold, 3-100 fold, 3-50 fold, 3-20 fold, 4-100 fold,4-50 fold, 4-40 fold, 4-30 fold, 4-25 fold, 4-20 fold, 4-15 fold, 4-10fold, 4-5 fold, 5-100 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold,5-20 fold, 5-15 fold, 5-10 fold, 6-100 fold, 6-50 fold, 6-40 fold, 6-30fold, 6-25 fold, 6-20 fold, 6-15 fold, 6-10 fold, 8-100 fold, 8-50 fold,8-40 fold, 8-30 fold, 8-25 fold, 8-20 fold, 8-15 fold, 8-10 fold, 10-100fold, 10-50 fold, 10-40 fold, 10-30 fold, 10-25 fold, 10-20 fold, 10-15fold, 20-100 fold, 20-50 fold, 20-40 fold, 20-30 fold, or 20-25 fold.

The disclosure provides lipid-comprising compounds and compositions thatare less susceptible to clearance and thus have a longer half-life invivo. This is particularly the case where the compositions are intendedfor repeated including chronic administration, and even moreparticularly where such repeated administration occurs within days orweeks.

Significantly, these compositions are less susceptible or altogethercircumvent the observed phenomenon of accelerated blood clearance (ABC).ABC is a phenomenon in which certain exogenously administered agents arerapidly cleared from the blood upon second and subsequentadministrations. This phenomenon has been observed, in part, for avariety of lipid-containing compositions including but not limited tolipidated agents, liposomes or other lipid-based delivery vehicles, andlipid-encapsulated agents. Heretofore, the basis of ABC has been poorlyunderstood and in some cases attributed to a humoral immune response andaccordingly strategies for limiting its impact in vivo particularly in aclinical setting have remained elusive.

This disclosure provides compounds and compositions that are lesssusceptible, if at all susceptible, to ABC. In some important aspects,such compounds and compositions are lipid-comprising compounds orcompositions. The lipid-containing compounds or compositions of thisdisclosure, surprisingly, do not experience ABC upon second andsubsequent administration in vivo. This resistance to ABC renders thesecompounds and compositions particularly suitable for repeated use invivo, including for repeated use within short periods of time, includingdays or 1-2 weeks. This enhanced stability and/or half-life is due, inpart, to the inability of these compositions to activate B1a and/or B1bcells and/or conventional B cells, pDCs and/or platelets.

This disclosure therefore provides an elucidation of the mechanismunderlying accelerated blood clearance (ABC). It has been found, inaccordance with this disclosure and the inventions provided herein, thatthe ABC phenomenon at least as it relates to lipids and lipidnanoparticles is mediated, at least in part an innate immune responseinvolving B1a and/or Bib cells, pDC and/or platelets. B1a cells arenormally responsible for secreting natural antibody, in the form ofcirculating IgM. This IgM is poly-reactive, meaning that it is able tobind to a variety of antigens, albeit with a relatively low affinity foreach.

It has been found in accordance with the invention that some lipidatedagents or lipid-comprising formulations such as lipid nanoparticlesadministered in vivo trigger and are subject to ABC. It has now beenfound in accordance with the invention that upon administration of afirst dose of the LNP, one or more cells involved in generating aninnate immune response (referred to herein as sensors) bind such agent,are activated, and then initiate a cascade of immune factors (referredto herein as effectors) that promote ABC and toxicity. For instance, B1aand B1b cells may bind to LNP, become activated (alone or in thepresence of other sensors such as pDC and/or effectors such as IL6) andsecrete natural IgM that binds to the LNP. Pre-existing natural IgM inthe subject may also recognize and bind to the LNP, thereby triggeringcomplement fixation. After administration of the first dose, theproduction of natural IgM begins within 1-2 hours of administration ofthe LNP. Typically by about 2-3 weeks the natural IgM is cleared fromthe system due to the natural half-life of IgM. Natural IgG is producedbeginning around 96 hours after administration of the LNP. The agent,when administered in a naïve setting, can exert its biological effectsrelatively unencumbered by the natural IgM produced post-activation ofthe B1a cells or B1b cells or natural IgG. The natural IgM and naturalIgG are non-specific and thus are distinct from anti-PEG IgM andanti-PEG IgG.

Although Applicant is not bound by mechanism, it is proposed that LNPstrigger ABC and/or toxicity through the following mechanisms. It isbelieved that when an LNP is administered to a subject the LNP israpidly transported through the blood to the spleen. The LNPs mayencounter immune cells in the blood and/or the spleen. A rapid innateimmune response is triggered in response to the presence of the LNPwithin the blood and/or spleen. Applicant has shown herein that withinhours of administration of an LNP several immune sensors have reacted tothe presence of the LNP. These sensors include but are not limited toimmune cells involved in generating an immune response, such as B cells,pDC, and platelets. The sensors may be present in the spleen, such as inthe marginal zone of the spleen and/or in the blood. The LNP mayphysically interact with one or more sensors, which may interact withother sensors. In such a case the LNP is directly or indirectlyinteracting with the sensors. The sensors may interact directly with oneanother in response to recognition of the LNP. For instance many sensorsare located in the spleen and can easily interact with one another.Alternatively one or more of the sensors may interact with LNP in theblood and become activated. The activated sensor may then interactdirectly with other sensors or indirectly (e.g., through the stimulationor production of a messenger such as a cytokine e.g., IL6).

In some embodiments the LNP may interact directly with and activate eachof the following sensors: pDC, B1a cells, B1b cells, and platelets.These cells may then interact directly or indirectly with one another toinitiate the production of effectors which ultimately lead to the ABCand/or toxicity associated with repeated doses of LNP. For instance,Applicant has shown that LNP administration leads to pDC activation,platelet aggregation and activation and B cell activation. In responseto LNP platelets also aggregate and are activated and aggregate with Bcells. pDC cells are activated. LNP has been found to interact with thesurface of platelets and B cells relatively quickly. Blocking theactivation of any one or combination of these sensors in response to LNPis useful for dampening the immune response that would ordinarily occur.This dampening of the immune response results in the avoidance of ABCand/or toxicity.

The sensors once activated produce effectors. An effector, as usedherein, is an immune molecule produced by an immune cell, such as a Bcell. Effectors include but are not limited to immunoglobulin such asnatural IgM and natural IgG and cytokines such as IL6. B1a and B1b cellsstimulate the production of natural IgMs within 2-6 hours followingadministration of an LNP. Natural IgG can be detected within 96 hours.IL6 levels are increased within several hours. The natural IgM and IgGcirculate in the body for several days to several weeks. During thistime the circulating effectors can interact with newly administeredLNPs, triggering those LNPs for clearance by the body. For instance, aneffector may recognize and bind to an LNP. The Fc region of the effectormay be recognized by and trigger uptake of the decorated LNP bymacrophage. The macrophage are then transported to the spleen. Theproduction of effectors by immune sensors is a transient response thatcorrelates with the timing observed for ABC.

If the administered dose is the second or subsequent administered dose,and if such second or subsequent dose is administered before thepreviously induced natural IgM and/or IgG is cleared from the system(e.g., before the 2-3 window time period), then such second orsubsequent dose is targeted by the circulating natural IgM and/ornatural IgG or Fc which trigger alternative complement pathwayactivation and is itself rapidly cleared. When LNP are administeredafter the effectors have cleared from the body or are reduced in number,ABC is not observed.

Thus, it is useful according to aspects of the invention to inhibit theinteraction between LNP and one or more sensors, to inhibit theactivation of one or more sensors by LNP (direct or indirect), toinhibit the production of one or more effectors, and/or to inhibit theactivity of one or more effectors. In some embodiments the LNP isdesigned to limit or block interaction of the LNP with a sensor. Forinstance the LNP may have an altered PC and/or PEG to preventinteractions with sensors. Alternatively or additionally an agent thatinhibits immune responses induced by LNPs may be used to achieve any oneor more of these effects.

It has also been determined that conventional B cells are alsoimplicated in ABC. Specifically, upon first administration of an agent,conventional B cells, referred to herein as CD19(+), bind to and reactagainst the agent. Unlike B1a and B1b cells though, conventional B cellsare able to mount first an IgM response (beginning around 96 hours afteradministration of the LNPs) followed by an IgG response (beginningaround 14 days after administration of the LNPs) concomitant with amemory response. Thus, conventional B cells react against theadministered agent and contribute to IgM (and eventually IgG) thatmediates ABC. The IgM and IgG are typically anti-PEG IgM and anti-PEGIgG.

It is contemplated that in some instances, the majority of the ABCresponse is mediated through B1a cells and B1a-mediated immuneresponses. It is further contemplated that in some instances, the ABCresponse is mediated by both IgM and IgG, with both conventional B cellsand B1a cells mediating such effects. In yet still other instances, theABC response is mediated by natural IgM molecules, some of which arecapable of binding to natural IgM, which may be produced by activatedB1a cells. The natural IgMs may bind to one or more components of theLNPs, e.g., binding to a phospholipid component of the LNPs (such asbinding to the PC moiety of the phospholipid) and/or binding to aPEG-lipid component of the LNPs (such as binding to PEG-DMG, inparticular, binding to the PEG moiety of PEG-DMG). Since B1a expressesCD36, to which phosphatidylcholine is a ligand, it is contemplated thatthe CD36 receptor may mediate the activation of B1a cells and thusproduction of natural IgM. In yet still other instances, the ABCresponse is mediated primarily by conventional B cells.

It has been found in accordance with the invention that the ABCphenomenon can be reduced or abrogated, at least in part, through theuse of compounds and compositions (such as agents, delivery vehicles,and formulations) that do not activate B1a cells. Compounds andcompositions that do not activate B1a cells may be referred to herein asB1a inert compounds and compositions. It has been further found inaccordance with the invention that the ABC phenomenon can be reduced orabrogated, at least in part, through the use of compounds andcompositions that do not activate conventional B cells. Compounds andcompositions that do not activate conventional B cells may in someembodiments be referred to herein as CD19-inert compounds andcompositions. Thus, in some embodiments provided herein, the compoundsand compositions do not activate B1a cells and they do not activateconventional B cells. Compounds and compositions that do not activateB1a cells and conventional B cells may in some embodiments be referredto herein as B1a/CD19-inert compounds and compositions.

These underlying mechanisms were not heretofore understood, and the roleof B1a and B1b cells and their interplay with conventional B cells inthis phenomenon was also not appreciated.

Accordingly, this disclosure provides compounds and compositions that donot promote ABC. These may be further characterized as not capable ofactivating B1a and/or B1b cells, platelets and/or pDC, and optionallyconventional B cells also. These compounds (e.g., agents, includingbiologically active agents such as prophylactic agents, therapeuticagents and diagnostic agents, delivery vehicles, including liposomes,lipid nanoparticles, and other lipid-based encapsulating structures,etc.) and compositions (e.g., formulations, etc.) are particularlydesirable for applications requiring repeated administration, and inparticular repeated administrations that occur within with short periodsof time (e.g., within 1-2 weeks). This is the case, for example, if theagent is a nucleic acid based therapeutic that is provided to a subjectat regular, closely-spaced intervals. The findings provided herein maybe applied to these and other agents that are similarly administeredand/or that are subject to ABC.

Of particular interest are lipid-comprising compounds, lipid-comprisingparticles, and lipid-comprising compositions as these are known to besusceptible to ABC. Such lipid-comprising compounds particles, andcompositions have been used extensively as biologically active agents oras delivery vehicles for such agents. Thus, the ability to improve theirefficacy of such agents, whether by reducing the effect of ABC on theagent itself or on its delivery vehicle, is beneficial for a widevariety of active agents.

Also provided herein are compositions that do not stimulate or boost anacute phase response (ARP) associated with repeat dose administration ofone or more biologically active agents.

The composition, in some instances, may not bind to IgM, including butnot limited to natural IgM.

The composition, in some instances, may not bind to an acute phaseprotein such as but not limited to C-reactive protein.

The composition, in some instances, may not trigger a CD5(+) mediatedimmune response. As used herein, a CD5(+) mediated immune response is animmune response that is mediated by B1a and/or B1b cells. Such aresponse may include an ABC response, an acute phase response, inductionof natural IgM and/or IgG, and the like.

The composition, in some instances, may not trigger a CD19(+) mediatedimmune response. As used herein, a CD19(+) mediated immune response isan immune response that is mediated by conventional CD19(+), CD5(−) Bcells. Such a response may include induction of IgM, induction of IgG,induction of memory B cells, an ABC response, an anti-drug antibody(ADA) response including an anti-protein response where the protein maybe encapsulated within an LNP, and the like.

B1a cells are a subset of B cells involved in innate immunity. Thesecells are the source of circulating IgM, referred to as natural antibodyor natural serum antibody. Natural IgM antibodies are characterized ashaving weak affinity for a number of antigens, and therefore they arereferred to as “poly-specific” or “poly-reactive”, indicating theirability to bind to more than one antigen. B1a cells are not able toproduce IgG. Additionally, they do not develop into memory cells andthus do not contribute to an adaptive immune response. However, they areable to secrete IgM upon activation. The secreted IgM is typicallycleared within about 2-3 weeks, at which point the immune system isrendered relatively naïve to the previously administered antigen. If thesame antigen is presented after this time period (e.g., at about 3 weeksafter the initial exposure), the antigen is not rapidly cleared.However, significantly, if the antigen is presented within that timeperiod (e.g., within 2 weeks, including within 1 week, or within days),then the antigen is rapidly cleared. This delay between consecutivedoses has rendered certain lipid-containing therapeutic or diagnosticagents unsuitable for use.

In humans, B1a cells are CD19(+), CD20(+), CD27(+), CD43(+), CD70(−) andCD5(+). In mice, B1a cells are CD19(+), CD5(+), and CD45 B cell isoformB220(+). It is the expression of CD5 which typically distinguishes B1acells from other convention B cells. B1a cells may express high levelsof CD5, and on this basis may be distinguished from other B-1 cells suchas B-1b cells which express low or undetectable levels of CD5. CD5 is apan-T cell surface glycoprotein. B1a cells also express CD36, also knownas fatty acid translocase. CD36 is a member of the class B scavengerreceptor family. CD36 can bind many ligands, including oxidized lowdensity lipoproteins, native lipoproteins, oxidized phospholipids, andlong-chain fatty acids.

B1b cells are another subset of B cells involved in innate immunity.These cells are another source of circulating natural IgM. Severalantigens, including PS, are capable of inducing T cell independentimmunity through B1b activation. CD27 is typically upregulated on B1bcells in response to antigen activation. Similar to B1a cells, the B1bcells are typically located in specific body locations such as thespleen and peritoneal cavity and are in very low abundance in the blood.The B1b secreted natural IgM is typically cleared within about 2-3weeks, at which point the immune system is rendered relatively naïve tothe previously administered antigen. If the same antigen is presentedafter this time period (e.g., at about 3 weeks after the initialexposure), the antigen is not rapidly cleared. However, significantly,if the antigen is presented within that time period (e.g., within 2weeks, including within 1 week, or within days), then the antigen israpidly cleared. This delay between consecutive doses has renderedcertain lipid-containing therapeutic or diagnostic agents unsuitable foruse.

In some embodiments it is desirable to block B1a and/or B1b cellactivation. One strategy for blocking B1a and/or B1b cell activationinvolves determining which components of a lipid nanoparticle promote Bcell activation and neutralizing those components. It has beendiscovered herein that at least PEG and phosphatidylcholine (PC)contribute to B1a and B1b cell interaction with other cells and/oractivation. PEG may play a role in promoting aggregation between B1cells and platelets, which may lead to activation. PC (a helper lipid inLNPs) is also involved in activating the B1 cells, likely throughinteraction with the CD36 receptor on the B cell surface. Numerousparticles have PEG-lipid alternatives, PEG-less, and/or PC replacementlipids (e.g. oleic acid or analogs thereof) have been designed andtested. Applicant has established that replacement of one or more ofthese components within an LNP that otherwise would promote ABC uponrepeat administration, is useful in preventing ABC by reducing theproduction of natural IgM and/or B cell activation. Thus, the inventionencompasses LNPs that have reduced ABC as a result of a design whicheliminates the inclusion of B cell triggers.

Another strategy for blocking B1a and/or B1b cell activation involvesusing an agent that inhibits immune responses induced by LNPs. Thesetypes of agents are discussed in more detail below. In some embodimentsthese agents block the interaction between B1a/B1b cells and the LNP orplatelets or pDC. For instance the agent may be an antibody or otherbinding agent that physically blocks the interaction. An example of thisis an antibody that binds to CD36 or CD6. The agent may also be acompound that prevents or disables the B1a/B1b cell from signaling onceactivated or prior to activation. For instance, it is possible to blockone or more components in the B1a/B1b signaling cascade the results fromB cell interaction with LNP or other immune cells. In other embodimentsthe agent may act one or more effectors produced by the B1a/B1b cellsfollowing activation. These effectors include for instance, natural IgMand cytokines.

It has been demonstrated according to aspects of the invention that whenactivation of pDC cells is blocked, B cell activation in response to LNPis decreased. Thus, in order to avoid ABC and/or toxicity, it may bedesirable to prevent pDC activation. Similar to the strategies discussedabove, pDC cell activation may be blocked by agents that interfere withthe interaction between pDC and LNP and/or B cells/platelets.Alternatively agents that act on the pDC to block its ability to getactivated or on its effectors can be used together with the LNP to avoidABC.

Platelets may also play an important role in ABC and toxicity. Veryquickly after a first dose of LNP is administered to a subject plateletsassociate with the LNP, aggregate and are activated. In some embodimentsit is desirable to block platelet aggregation and/or activation. Onestrategy for blocking platelet aggregation and/or activation involvesdetermining which components of a lipid nanoparticle promote plateletaggregation and/or activation and neutralizing those components. It hasbeen discovered herein that at least PEG contribute to plateletaggregation, activation and/or interaction with other cells. Numerousparticles have PEG-lipid alternatives and PEG-less have been designedand tested. Applicant has established that replacement of one or more ofthese components within an LNP that otherwise would promote ABC uponrepeat administration, is useful in preventing ABC by reducing theproduction of natural IgM and/or platelet aggregation. Thus, theinvention encompasses LNPs that have reduced ABC as a result of a designwhich eliminates the inclusion of platelet triggers. Alternativelyagents that act on the platelets to block its activity once it isactivated or on its effectors can be used together with the LNP to avoidABC.

(i) Measuring ABC Activity and Related Activities

Various compounds and compositions provided herein, including LNPs, donot promote ABC activity upon administration in vivo. These LNPs may becharacterized and/or identified through any of a number of assays, suchas but not limited to those described below, as well as any of theassays disclosed in the Examples section, include the methods subsectionof the Examples.

In some embodiments the methods involve administering an LNP withoutproducing an immune response that promotes ABC. An immune response thatpromotes ABC involves activation of one or more sensors, such as B1cells, pDC, or platelets, and one or more effectors, such as naturalIgM, natural IgG or cytokines such as IL6. Thus administration of an LNPwithout producing an immune response that promotes ABC, at a minimuminvolves administration of an LNP without significant activation of oneor more sensors and significant production of one or more effectors.Significant used in this context refers to an amount that would lead tothe physiological consequence of accelerated blood clearance of all orpart of a second dose with respect to the level of blood clearanceexpected for a second dose of an ABC triggering LNP. For instance, theimmune response should be dampened such that the ABC observed after thesecond dose is lower than would have been expected for an ABC triggeringLNP.

(ii) B1a or B1b Activation Assay

Certain compositions provided in this disclosure do not activate Bcells, such as B1a or B1b cells (CD19+CD5+) and/or conventional B cells(CD19+CD5−). Activation of B1a cells, B1b cells, or conventional B cellsmay be determined in a number of ways, some of which are provided below.B cell population may be provided as fractionated B cell populations orunfractionated populations of splenocytes or peripheral bloodmononuclear cells (PBMC). If the latter, the cell population may beincubated with the LNP of choice for a period of time, and thenharvested for further analysis. Alternatively, the supernatant may beharvested and analyzed.

(iii) Upregulation of Activation Marker Cell Surface Expression

Activation of B1a cells, B1b cells, or conventional B cells may bedemonstrated as increased expression of B cell activation markersincluding late activation markers such as CD86. In an exemplarynon-limiting assay, unfractionated B cells are provided as a splenocytepopulation or as a PBMC population, incubated with an LNP of choice fora particular period of time, and then stained for a standard B cellmarker such as CD19 and for an activation marker such as CD86, andanalyzed using for example flow cytometry. A suitable negative controlinvolves incubating the same population with medium, and then performingthe same staining and visualization steps. An increase in CD86expression in the test population compared to the negative controlindicates B cell activation.

(iv) Pro-Inflammatory Cytokine Release

B cell activation may also be assessed by cytokine release assay. Forexample, activation may be assessed through the production and/orsecretion of cytokines such as IL-6 and/or TNF-alpha upon exposure withLNPs of interest.

Such assays may be performed using routine cytokine secretion assayswell known in the art. An increase in cytokine secretion is indicativeof B cell activation.

(v) LNP Binding/Association to and/or Uptake by B Cells

LNP association or binding to B cells may also be used to assess an LNPof interest and to further characterize such LNP. Association/bindingand/or uptake/internalization may be assessed using a detectablylabeled, such as fluorescently labeled, LNP and tracking the location ofsuch LNP in or on B cells following various periods of incubation.

The invention further contemplates that the compositions provided hereinmay be capable of evading recognition or detection and optionallybinding by downstream mediators of ABC such as circulating IgM and/oracute phase response mediators such as acute phase proteins (e.g.,C-reactive protein (CRP).

(vi) Methods of Use for Reducing ABC

Also provided herein are methods for delivering LNPs, which mayencapsulate an agent such as a therapeutic agent, to a subject withoutpromoting ABC.

In some embodiments, the method comprises administering any of the LNPsdescribed herein, which do not promote ABC, for example, do not induceproduction of natural IgM binding to the LNPs, do not activate B1aand/or B1b cells. As used herein, an LNP that “does not promote ABC”refers to an LNP that induces no immune responses that would lead tosubstantial ABC or a substantially low level of immune responses that isnot sufficient to lead to substantial ABC. An LNP that does not inducethe production of natural IgMs binding to the LNP refers to LNPs thatinduce either no natural IgM binding to the LNPs or a substantially lowlevel of the natural IgM molecules, which is insufficient to lead tosubstantial ABC. An LNP that does not activate B1a and/or B1b cellsrefer to LNPs that induce no response of B1a and/or B1b cells to producenatural IgM binding to the LNPs or a substantially low level of B1aand/or B1b responses, which is insufficient to lead to substantial ABC.

In some embodiments the terms do not activate and do not induceproduction are a relative reduction to a reference value or condition.In some embodiments the reference value or condition is the amount ofactivation or induction of production of a molecule such as IgM by astandard LNP such as an MC3 LNP. In some embodiments the relativereduction is a reduction of at least 30%, for example at least 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments the terms donot activate cells such as B cells and do not induce production of aprotein such as IgM may refer to an undetectable amount of the activecells or the specific protein.

(vii) Platelet Effects and Toxicity

The invention is further premised in part on the elucidation of themechanism underlying dose-limiting toxicity associated with LNPadministration. Such toxicity may involve coagulopathy, disseminatedintravascular coagulation (DIC, also referred to as consumptivecoagulopathy), whether acute or chronic, and/or vascular thrombosis. Insome instances, the dose-limiting toxicity associated with LNPs is acutephase response (APR) or complement activation-related psudoallergy(CARPA).

As used herein, coagulopathy refers to increased coagulation (bloodclotting) in vivo. The findings reported in this disclosure areconsistent with such increased coagulation and significantly provideinsight on the underlying mechanism. Coagulation is a process thatinvolves a number of different factors and cell types, and heretoforethe relationship between and interaction of LNPs and platelets has notbeen understood in this regard. This disclosure provides evidence ofsuch interaction and also provides compounds and compositions that aremodified to have reduced platelet effect, including reduced plateletassociation, reduced platelet aggregation, and/or reduced plateletaggregation. The ability to modulate, including preferablydown-modulate, such platelet effects can reduce the incidence and/orseverity of coagulopathy post-LNP administration. This in turn willreduce toxicity relating to such LNP, thereby allowing higher doses ofLNPs and importantly their cargo to be administered to patients in needthereof.

CARPA is a class of acute immune toxicity manifested in hypersensitivityreactions (HSRs), which may be triggered by nanomedicines andbiologicals. Unlike allergic reactions, CARPA typically does not involveIgE but arises as a consequence of activation of the complement system,which is part of the innate immune system that enhances the body'sabilities to clear pathogens. One or more of the following pathways, theclassical complement pathway (CP), the alternative pathway (AP), and thelectin pathway (LP), may be involved in CARPA. Szebeni, MolecularImmunology, 61:163-173 (2014).

The classical pathway is triggered by activation of the C1-complex,which contains. C1q, C1r, C1s, or C1qr2s2. Activation of the C1-complexoccurs when C1q binds to IgM or IgG complexed with antigens, or when C1qbinds directly to the surface of the pathogen. Such binding leads toconformational changes in the C1q molecule, which leads to theactivation of C1r, which in turn, cleave C1s. The C1r2s2 component nowsplits C4 and then C2, producing C4a, C4b, C2a, and C2b. C4b and C2bbind to form the classical pathway C3-convertase (C4b2b complex), whichpromotes cleavage of C3 into C3a and C3b. C3b then binds the C3convertase to from the C5 convertase (C4b2b3b complex). The alternativepathway is continuously activated as a result of spontaneous C3hydrolysis. Factor P (properdin) is a positive regulator of thealternative pathway. Oligomerization of properdin stabilizes the C3convertase, which can then cleave much more C3. The C3 molecules canbind to surfaces and recruit more B, D, and P activity, leading toamplification of the complement activation.

Acute phase response (APR) is a complex systemic innate immune responsesfor preventing infection and clearing potential pathogens. Numerousproteins are involved in APR and C-reactive protein is awell-characterized one.

It has been found, in accordance with the invention, that certain LNPare able to associate physically with platelets almost immediately afteradministration in vivo, while other LNP do not associate with plateletsat all or only at background levels. Significantly, those LNPs thatassociate with platelets also apparently stabilize the plateletaggregates that are formed thereafter. Physical contact of the plateletswith certain LNPs correlates with the ability of such platelets toremain aggregated or to form aggregates continuously for an extendedperiod of time after administration. Such aggregates comprise activatedplatelets and also innate immune cells such as macrophages and B cells.

25. Methods of Use

The polynucleotides, pharmaceutical compositions and formulationsdescribed herein are used in the preparation, manufacture andtherapeutic use of to treat and/or prevent Citrin-related diseases,disorders or conditions. In some embodiments, the polynucleotides,compositions and formulations of the invention are used to treat and/orprevent CTLN2.

In some embodiments, the polynucleotides, pharmaceutical compositionsand formulations of the invention are used in methods for reducingammonia levels and/or reducing triglyceride levels in a subject in needthereof. For instance, one aspect of the invention provides a method ofalleviating the signs and symptoms of CTLN2 in a subject comprising theadministration of a composition or formulation comprising apolynucleotide encoding Citrin to that subject (e.g., an mRNA encoding aCitrin polypeptide).

In some embodiments, the polynucleotides, pharmaceutical compositionsand formulations of the invention are used to reduce the level of ametabolite associated with CTLN2 (e.g., the substrate or product, i.e.,glutamate, aspartate, or ammonia), the method comprising administeringto the subject an effective amount of a polynucleotide encoding a Citrinpolypeptide.

In some embodiments, the administration of an effective amount of apolynucleotide, pharmaceutical composition or formulation of theinvention reduces the levels of a biomarker of CTLN2, e.g., glutamate,aspartate, or ammonia. In some embodiments, the administration of thepolynucleotide, pharmaceutical composition or formulation of theinvention results in reduction in the level of one or more biomarkers ofCTLN2, e.g., ammonia, triglycerides, glutamate, aspartate, citrulline,pyruvate and lactate, within a short period of time after administrationof the polynucleotide, pharmaceutical composition or formulation of theinvention.

In some embodiments, the level of one or more biomarkers of CTLN2, e.g.,ammonia, triglycerides, glutamate, aspartate, citrulline, pyruvate andlactate, is measured in blood. In some embodiments, the level of one ofmore biomarkers is measured in a component of blood, for example inplasma or serum. Methods of obtaining blood and components of blood andfor measuring the level of biomarkers in blood or a component of bloodare known in the art. In some embodiments, the level of one or morebiomarkers of CTLN2, e.g., ammonia, glutamate, aspartate, citrulline,pyruvate and lactate, is measured in urine. Methods of obtaining urineand for measuring the level of biomarkers in urine are known in the art.In some embodiments, the level of one or more biomarkers of CTLN2, e.g.,ammonia, triglycerides, glutamate, aspartate, citrulline, pyruvate andlactate, is measured in liver tissue. Methods of obtaining liver tissue,e.g. biopsy, and for measuring the level of biomarkers in liver tissueare known in the art.

In some embodiments, the level of one or more biomarkers of CTLN2, e.g.,ammonia, triglycerides, glutamate, aspartate, citrulline, pyruvate andlactate, is measured in a subject to determine the change in level ofthe biomarker after administration of a pharmaceutical composition orpolynucleotide as described herein. In some embodiments, the level ofthe biomarker may be reduced at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, atleast 15 fold, at least 20-fold, at least 25-fold, at least 30-fold, atleast 35-fold, at least 40-fold, at least 45-fold or at least 50-foldcompared to a reference biomarker level in a subject having CTLN2.

In some embodiments, the blood, plasma or serum level of ammonia isreduced at least 2-fold, at least 3-fold, at least 5-fold, at least10-fold, at least 15 fold, at least 20-fold, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% or at least 98%, at least 99%, or 100% comparedto a reference ammonia blood, plasma or serum level in a subject havingCTLN2, for at least 24 hours, at least 48 hours, at least 72 hours, atleast 96 hours, or at least 120 hours post-administration of apharmaceutical composition or polynucleotide as described herein.Reference levels of ammonia in the blood, plasma or serum or subjectshaving CTLN2 and in subjects that do not have CTLN2 can be found in theart, for example, in Kobayashi et al., Citrin Deficiency, GeneReviews2005 (available at https://www.ncbi.nlm.nih.gov/books/NBK1181/) and inWong et al., Arch Neurol 64(7):1034-1037 (2007), both of which arehereby incorporated by reference herein.

Replacement therapy is a potential treatment for CTLN2. Thus, in certainaspects of the invention, the polynucleotides, e.g., mRNA, disclosedherein comprise one or more sequences encoding a Citrin polypeptide thatis suitable for use in gene replacement therapy for CTLN2. In someembodiments, the present disclosure treats a lack of or reduced Citrinexpression and/or Citrin activity, or decreased or abnormal Citrinactivity in a subject by providing a polynucleotide, e.g., mRNA, thatencodes a Citrin polypeptide to the subject. In some embodiments, thepolynucleotide is sequence-optimized. In some embodiments, thepolynucleotide (e.g., an mRNA) comprises a nucleic acid sequence (e.g.,an ORF) encoding a Citrin polypeptide, wherein the nucleic acid issequence-optimized, e.g., by modifying its G/C, uridine, or thymidinecontent, and/or the polynucleotide comprises at least one chemicallymodified nucleoside. In some embodiments, the polynucleotide comprises amiRNA binding site, e.g., a miRNA binding site that binds miRNA-142,miRNA-126, or a combination thereof.

In some embodiments, the administration of a composition or formulationcomprising polynucleotide, pharmaceutical composition or formulation ofthe invention to a subject results in an increase in mitochondrialuptake of glutamate, increase in mitochondrial export of aspartateand/or decrease in ammonia to a level at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or to 100% lower than the level observed prior to theadministration of the composition or formulation.

In some embodiments, the administration of the polynucleotide,pharmaceutical composition or formulation of the invention results inexpression of Citrin in cells of the subject. In some embodiments,administering the polynucleotide, pharmaceutical composition orformulation of the invention results in an increase of Citrin expressionand/or activity in the subject. For example, in some embodiments, thepolynucleotides of the present invention are used in methods ofadministering a composition or formulation comprising an mRNA encoding aCitrin polypeptide to a subject, wherein the method results in anincrease of Citrin expression and/or activity in at least some cells ofa subject.

In some embodiments, the administration of a composition or formulationcomprising an mRNA encoding a Citrin polypeptide to a subject results inan increase of Citrin expression and/or activity in cells subject to alevel at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or to 100% or more of theexpression and/or activity level expected in a normal subject, e.g., ahuman not suffering from CTLN2.

In some embodiments, the administration of the polynucleotide,pharmaceutical composition or formulation of the invention results inexpression of Citrin protein in at least some of the cells of a subjectthat persists for a period of time sufficient to allow reduction ofammonia to occur.

In some embodiments, the expression and/or activity of the encodedpolypeptide is increased. In some embodiments, the polynucleotideincreases Citrin expression levels in cells when introduced into thosecells, e.g., by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or to 100% withrespect to the Citrin expression and/or activity level in the cellsbefore the polypeptide is introduced in the cells.

In some embodiments, the method or use comprises administering apolynucleotide, e.g., mRNA, comprising a nucleotide sequence havingsequence similarity to a polynucleotide selected from the group of SEQID NOs: 5-29, 129, 132, 135, and 138 (See TABLE 2), or a polynucleotideselected from the group of SEQ ID NOs: 130, 133, 136, and 139 (See TABLE5), wherein the polynucleotide encodes an Citrin polypeptide.

Other aspects of the present disclosure relate to transplantation ofcells containing polynucleotides to a mammalian subject. Administrationof cells to mammalian subjects is known to those of ordinary skill inthe art, and includes, but is not limited to, local implantation (e.g.,topical or subcutaneous administration), organ delivery or systemicinjection (e.g., intravenous injection or inhalation), and theformulation of cells in pharmaceutically acceptable carriers.

Also provided is a method to increase survival an CTLN2 patientcomprising administering to the subject a therapeutically effectiveamount of a composition or formulation comprising mRNA encoding a Citrinpolypeptide disclosed herein.

In some aspects, the dose of mRNA encoding a Citrin polypeptidedisclosed herein is at least about 0.1 nmol/kg, at least about 0.2nmol/kg, at least about 0.3 nmol/kg, at least about 0.4 nmol/kg, atleast about 0.5 nmol/kg, at least about 0.6 nmol/kg, at least about 0.7nmol/kg, at least about 0.8 nmol/kg, at least about 0.9 nmol/kg, atleast about 1 nmol/kg, at least about 1.1 nmol/kg, at least about 1.2nmol/kg, at least about 1.3 nmol/kg, at least about 1.4 nmol/kg, atleast about 1.5 nmol/kg, at least about 1.6 nmol/kg, at least about 1.7nmol/kg, at least about 1.8 nmol/kg, at least about 1.9 nmol/kg, atleast about 2 nmol/kg, at least about 2.5 nmol/kg, at least about 3nmol/kg, at least about 3.5 nmol/kg, at least about 4 nmol/kg, at leastabout 4.5 nmol/kg, or at least about 5 nmol/kg. In some aspects, thedose of mRNA encoding a Citrin polypeptide disclosed herein is at leastabout 0.05 mg/kg, at least about 0.1 mg/kg, at least about 0.15 mg/kg,at least about 0.2 mg/kg, at least about 0.25 mg/kg, at least about 0.3mg/kg, at least about 0.35 mg/kg, at least about 0.4 mg/kg, at leastabout 0.45 mg/kg, at least about 0.5 mg/kg, at least about 0.55 mg/kg,at least about 0.6 mg/kg, at least about 0.7 mg/kg, at least about 0.75mg/kg, at least about 0.8 mg/kg, at least about 0.85 mg/kg, at leastabout 0.9 mg/kg, at least about 0.95 mg/kg, or at least about 1 mg/kg.

In some aspects of the method disclosed herein, the CTLN2 patient is anasymptomatic patient.

In some embodiments, the polynucleotides (e.g., mRNA), pharmaceuticalcompositions and formulations used in the methods of the inventioncomprise a uracil-modified sequence encoding a Citrin polypeptidedisclosed herein and a miRNA binding site disclosed herein, e.g., amiRNA binding site that binds to miR-142 and/or a miRNA binding sitethat binds to miR-126. In some embodiments, the uracil-modified sequenceencoding a Citrin polypeptide comprises at least one chemically modifiednucleobase, e.g., 5-methoxyuracil. In some embodiments, at least 95% ofa type of nucleobase (e.g., uracil) in a uracil-modified sequenceencoding a Citrin polypeptide of the invention are modified nucleobases.In some embodiments, at least 95% of uricil in a uracil-modifiedsequence encoding a Citrin polypeptide is 5-methoxyuridine. In someembodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) disclosedherein is formulated with a delivery agent comprising, e.g., a compoundhaving the Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18;a compound having the Formula (III), (IV), (V), or (VI), e.g., any ofCompounds 233-342, e.g., Compound 236; or a compound having the Formula(VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or anycombination thereof. In some embodiments, the delivery agent comprisesCompound 18, DSPC, Cholesterol, and Compound 428, e.g., with a moleratio of about 50:10:38.5:1.5.

The skilled artisan will appreciate that the therapeutic effectivenessof a drug or a treatment of the instant invention can be characterizedor determined by measuring the level of expression of an encoded protein(e.g., enzyme) in a sample or in samples taken from a subject (e.g.,from a preclinical test subject (rodent, primate, etc.) or from aclinical subject (human). Likewise, the therapeutic effectiveness of adrug or a treatment of the instant invention can be characterized ordetermined by measuring the level of activity of an encoded protein(e.g., enzyme) in a sample or in samples taken from a subject (e.g.,from a preclinical test subject (rodent, primate, etc.) or from aclinical subject (human). Furthermore, the therapeutic effectiveness ofa drug or a treatment of the instant invention can be characterized ordetermined by measuring the level of an appropriate biomarker insample(s) taken from a subject. Levels of protein and/or biomarkers canbe determined post-administration with a single dose of an mRNAtherapeutic of the invention or can be determined and/or monitored atseveral time points following administration with a single dose or canbe determined and/or monitored throughout a course of treatment, e.g., amulti-dose treatment.

26. Compositions and Formulations for Use

Certain aspects of the invention are directed to compositions orformulations comprising any of the polynucleotides disclosed above.

In some embodiments, the composition or formulation comprises: (i) apolynucleotide (e.g., a RNA, e.g., an mRNA) comprising asequence-optimized nucleotide sequence (e.g., an ORF) encoding a Citrinpolypeptide (e.g., the wild-type sequence, functional fragment, orvariant thereof), wherein the polynucleotide comprises at least onechemically modified nucleobase, e.g., 5-methoxyuracil (e.g., wherein atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 99%, or 100% of theuracils are 5-methoxyuracils), and wherein the polynucleotide furthercomprises a miRNA binding site, e.g., a miRNA binding site that binds tomiR-142 (e.g., a miR-142-3p or miR-142-5p binding site); and (ii) adelivery agent comprising, e.g., a compound having the Formula (I),e.g., any of Compounds 1-232, e.g., Compound 18; a compound having theFormula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g.,Compound 236; or a compound having the Formula (VIII), e.g., any ofCompounds 419-428, e.g., Compound 428, or any combination thereof. Insome embodiments, the delivery agent comprises Compound 18, DSPC,Cholesterol, and Compound 428, e.g., with a mole ratio of about50:10:38.5:1.5.

In some embodiments, the uracil or thymine content of the ORF relativeto the theoretical minimum uracil or thymine content of a nucleotidesequence encoding the Citrin polypeptide (% U_(TM) or % T_(TM)), isbetween about 100% and about 150%.

In some embodiments, the polynucleotides, compositions or formulationsabove are used to treat and/or prevent a Citrin-related diseases,disorders or conditions, e.g., CTLN2.

27. Forms of Administration

The polynucleotides, pharmaceutical compositions and formulations of theinvention described above can be administered by any route that resultsin a therapeutically effective outcome. These include, but are notlimited to enteral (into the intestine), gastroenteral, epidural (intothe dura matter), oral (by way of the mouth), transdermal, peridural,intracerebral (into the cerebrum), intracerebroventricular (into thecerebral ventricles), epicutaneous (application onto the skin),intradermal, (into the skin itself), subcutaneous (under the skin),nasal administration (through the nose), intravenous (into a vein),intravenous bolus, intravenous drip, intraarterial (into an artery),intramuscular (into a muscle), intracardiac (into the heart),intraosseous infusion (into the bone marrow), intrathecal (into thespinal canal), intraperitoneal, (infusion or injection into theperitoneum), intravesical infusion, intravitreal, (through the eye),intracavernous injection (into a pathologic cavity) intracavitary (intothe base of the penis), intravaginal administration, intrauterine,extra-amniotic administration, transdermal (diffusion through the intactskin for systemic distribution), transmucosal (diffusion through amucous membrane), transvaginal, insufflation (snorting), sublingual,sublabial, enema, eye drops (onto the conjunctiva), in ear drops,auricular (in or by way of the ear), buccal (directed toward the cheek),conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis,endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis,infiltration, interstitial, intra-abdominal, intra-amniotic,intra-articular, intrabiliary, intrabronchial, intrabursal,intracartilaginous (within a cartilage), intracaudal (within the caudaequine), intracisternal (within the cisterna magna cerebellomedularis),intracorneal (within the cornea), dental intracornal, intracoronary(within the coronary arteries), intracorporus cavernosum (within thedilatable spaces of the corporus cavernosa of the penis), intradiscal(within a disc), intraductal (within a duct of a gland), intraduodenal(within the duodenum), intradural (within or beneath the dura),intraepidermal (to the epidermis), intraesophageal (to the esophagus),intragastric (within the stomach), intragingival (within the gingivae),intraileal (within the distal portion of the small intestine),intralesional (within or introduced directly to a localized lesion),intraluminal (within a lumen of a tube), intralymphatic (within thelymph), intramedullary (within the marrow cavity of a bone),intrameningeal (within the meninges), intraocular (within the eye),intraovarian (within the ovary), intrapericardial (within thepericardium), intrapleural (within the pleura), intraprostatic (withinthe prostate gland), intrapulmonary (within the lungs or its bronchi),intrasinal (within the nasal or periorbital sinuses), intraspinal(within the vertebral column), intrasynovial (within the synovial cavityof a joint), intratendinous (within a tendon), intratesticular (withinthe testicle), intrathecal (within the cerebrospinal fluid at any levelof the cerebrospinal axis), intrathoracic (within the thorax),intratubular (within the tubules of an organ), intratympanic (within theaurus media), intravascular (within a vessel or vessels),intraventricular (within a ventricle), iontophoresis (by means ofelectric current where ions of soluble salts migrate into the tissues ofthe body), irrigation (to bathe or flush open wounds or body cavities),laryngeal (directly upon the larynx), nasogastric (through the nose andinto the stomach), occlusive dressing technique (topical routeadministration that is then covered by a dressing that occludes thearea), ophthalmic (to the external eye), oropharyngeal (directly to themouth and pharynx), parenteral, percutaneous, periarticular, peridural,perineural, periodontal, rectal, respiratory (within the respiratorytract by inhaling orally or nasally for local or systemic effect),retrobulbar (behind the pons or behind the eyeball), intramyocardial(entering the myocardium), soft tissue, subarachnoid, subconjunctival,submucosal, topical, transplacental (through or across the placenta),transtracheal (through the wall of the trachea), transtympanic (acrossor through the tympanic cavity), ureteral (to the ureter), urethral (tothe urethra), vaginal, caudal block, diagnostic, nerve block, biliaryperfusion, cardiac perfusion, photopheresis or spinal. In specificembodiments, compositions can be administered in a way that allows themcross the blood-brain barrier, vascular barrier, or other epithelialbarrier. In some embodiments, a formulation for a route ofadministration can include at least one inactive ingredient.

The polynucleotides of the present invention (e.g., a polynucleotidecomprising a nucleotide sequence encoding a Citrin polypeptide or afunctional fragment or variant thereof) can be delivered to a cellnaked. As used herein in, “naked” refers to delivering polynucleotidesfree from agents that promote transfection. The naked polynucleotidescan be delivered to the cell using routes of administration known in theart and described herein.

The polynucleotides of the present invention (e.g., a polynucleotidecomprising a nucleotide sequence encoding a Citrin polypeptide or afunctional fragment or variant thereof) can be formulated, using themethods described herein. The formulations can contain polynucleotidesthat can be modified and/or unmodified. The formulations can furtherinclude, but are not limited to, cell penetration agents, apharmaceutically acceptable carrier, a delivery agent, a bioerodible orbiocompatible polymer, a solvent, and a sustained-release deliverydepot. The formulated polynucleotides can be delivered to the cell usingroutes of administration known in the art and described herein.

A pharmaceutical composition for parenteral administration can compriseat least one inactive ingredient. Any or none of the inactiveingredients used can have been approved by the US Food and DrugAdministration (FDA). A non-exhaustive list of inactive ingredients foruse in pharmaceutical compositions for parenteral administrationincludes hydrochloric acid, mannitol, nitrogen, sodium acetate, sodiumchloride and sodium hydroxide.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations can be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables. The sterileformulation can also comprise adjuvants such as local anesthetics,preservatives and buffering agents.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Injectable formulations can be for direct injection into a region of atissue, organ and/or subject. As a non-limiting example, a tissue, organand/or subject can be directly injected a formulation by intramyocardialinjection into the ischemic region. (See, e.g., Zangi et al. NatureBiotechnology 2013; the contents of which are herein incorporated byreference in its entirety).

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, can depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

28. Kits and Devices

a. Kits

The invention provides a variety of kits for conveniently and/oreffectively using the claimed nucleotides of the present invention.Typically kits will comprise sufficient amounts and/or numbers ofcomponents to allow a user to perform multiple treatments of asubject(s) and/or to perform multiple experiments.

In one aspect, the present invention provides kits comprising themolecules (polynucleotides) of the invention.

Said kits can be for protein production, comprising a firstpolynucleotides comprising a translatable region. The kit can furthercomprise packaging and instructions and/or a delivery agent to form aformulation composition. The delivery agent can comprise a saline, abuffered solution, a lipidoid or any delivery agent disclosed herein.

In some embodiments, the buffer solution can include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution can include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium and mannose (See, e.g., U.S. Pub. No.20120258046; herein incorporated by reference in its entirety). In afurther embodiment, the buffer solutions can be precipitated or it canbe lyophilized. The amount of each component can be varied to enableconsistent, reproducible higher concentration saline or simple bufferformulations. The components can also be varied in order to increase thestability of modified RNA in the buffer solution over a period of timeand/or under a variety of conditions. In one aspect, the presentinvention provides kits for protein production, comprising: apolynucleotide comprising a translatable region, provided in an amounteffective to produce a desired amount of a protein encoded by thetranslatable region when introduced into a target cell; a secondpolynucleotide comprising an inhibitory nucleic acid, provided in anamount effective to substantially inhibit the innate immune response ofthe cell; and packaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide comprising a translatableregion, wherein the polynucleotide exhibits reduced degradation by acellular nuclease, and packaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide comprising a translatableregion, wherein the polynucleotide exhibits reduced degradation by acellular nuclease, and a mammalian cell suitable for translation of thetranslatable region of the first nucleic acid.

b. Devices

The present invention provides for devices that can incorporatepolynucleotides that encode polypeptides of interest. These devicescontain in a stable formulation the reagents to synthesize apolynucleotide in a formulation available to be immediately delivered toa subject in need thereof, such as a human patient

Devices for administration can be employed to deliver thepolynucleotides of the present invention according to single, multi- orsplit-dosing regimens taught herein. Such devices are taught in, forexample, International Application Publ. No. WO2013151666, the contentsof which are incorporated herein by reference in their entirety.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentinvention. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present invention, these multi-administration devicescan be utilized to deliver the single, multi- or split dosescontemplated herein. Such devices are taught for example in,International Application Publ. No. WO2013151666, the contents of whichare incorporated herein by reference in their entirety.

In some embodiments, the polynucleotide is administered subcutaneouslyor intramuscularly via at least 3 needles to three different, optionallyadjacent, sites simultaneously, or within a 60 minutes period (e.g.,administration to 4, 5, 6, 7, 8, 9, or 10 sites simultaneously or withina 60 minute period).

c. Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens can be employed toadminister the polynucleotides of the present invention on a single,multi- or split dosing schedule. Such methods and devices are describedin International Application Publication No. WO2013151666, the contentsof which are incorporated herein by reference in their entirety.

d. Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current can be employed todeliver the polynucleotides of the present invention according to thesingle, multi- or split dosing regimens taught herein. Such methods anddevices are described in International Application Publication No.WO2013151666, the contents of which are incorporated herein by referencein their entirety.

29. Definitions

In order that the present disclosure can be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The invention includes embodiments in which exactly one member of thegroup is present in, employed in, or otherwise relevant to a givenproduct or process. The invention includes embodiments in which morethan one, or all of the group members are present in, employed in, orotherwise relevant to a given product or process.

In this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlydictates otherwise. The terms “a” (or “an”), as well as the terms “oneor more,” and “at least one” can be used interchangeably herein. Incertain aspects, the term “a” or “an” means “single.” In other aspects,the term “a” or “an” includes “two or more” or “multiple.”

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Wherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Where a range of values is recited, it is tobe understood that each intervening integer value, and each fractionthereof, between the recited upper and lower limits of that range isalso specifically disclosed, along with each subrange between suchvalues. The upper and lower limits of any range can independently beincluded in or excluded from the range, and each range where either,neither or both limits are included is also encompassed within theinvention. Where a value is explicitly recited, it is to be understoodthat values which are about the same quantity or amount as the recitedvalue are also within the scope of the invention. Where a combination isdisclosed, each subcombination of the elements of that combination isalso specifically disclosed and is within the scope of the invention.Conversely, where different elements or groups of elements areindividually disclosed, combinations thereof are also disclosed. Whereany element of an invention is disclosed as having a plurality ofalternatives, examples of that invention in which each alternative isexcluded singly or in any combination with the other alternatives arealso hereby disclosed; more than one element of an invention can havesuch exclusions, and all combinations of elements having such exclusionsare hereby disclosed.

Nucleotides are referred to by their commonly accepted single-lettercodes. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation. Nucleobases are referred to herein bytheir commonly known one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Accordingly, A represents adenine,C represents cytosine, G represents guanine, T represents thymine, Urepresents uracil.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Unless otherwise indicated, aminoacid sequences are written left to right in amino to carboxyorientation.

About: The term “about” as used in connection with a numerical valuethroughout the specification and the claims denotes an interval ofaccuracy, familiar and acceptable to a person skilled in the art. Suchinterval of accuracy is ±10%.

Where ranges are given, endpoints are included. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there can be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Amino acid substitution: The term “amino acid substitution” refers toreplacing an amino acid residue present in a parent or referencesequence (e.g., a wild type Citrin sequence) with another amino acidresidue. An amino acid can be substituted in a parent or referencesequence (e.g., a wild type Citrin polypeptide sequence), for example,via chemical peptide synthesis or through recombinant methods known inthe art. Accordingly, a reference to a “substitution at position X”refers to the substitution of an amino acid present at position X withan alternative amino acid residue. In some aspects, substitutionpatterns can be described according to the schema AnY, wherein A is thesingle letter code corresponding to the amino acid naturally ororiginally present at position n, and Y is the substituting amino acidresidue. In other aspects, substitution patterns can be describedaccording to the schema An(YZ), wherein A is the single letter codecorresponding to the amino acid residue substituting the amino acidnaturally or originally present at position X, and Y and Z arealternative substituting amino acid residues.

In the context of the present disclosure, substitutions (even when theyreferred to as amino acid substitution) are conducted at the nucleicacid level, i.e., substituting an amino acid residue with an alternativeamino acid residue is conducted by substituting the codon encoding thefirst amino acid with a codon encoding the second amino acid.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Approximately: As used herein, the term “approximately,” as applied toone or more values of interest, refers to a value that is similar to astated reference value. In certain embodiments, the term “approximately”refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,or less in either direction (greater than or less than) of the statedreference value unless otherwise stated or otherwise evident from thecontext (except where such number would exceed 100% of a possiblevalue).

Associated with: As used herein with respect to a disease, the term“associated with” means that the symptom, measurement, characteristic,or status in question is linked to the diagnosis, development, presence,or progression of that disease. As association can, but need not, becausatively linked to the disease. For example, signs, symptoms,sequelae, or any effects causing a decrease in the quality of life of apatient of CTLN2 are considered associated with CTLN2 and in someembodiments of the present invention can be treated, ameliorated, orprevented by administering the polynucleotides of the present inventionto a subject in need thereof.

When used with respect to two or more moieties, the terms “associatedwith,” “conjugated,” “linked,” “attached,” and “tethered,” when usedwith respect to two or more moieties, means that the moieties arephysically associated or connected with one another, either directly orvia one or more additional moieties that serves as a linking agent, toform a structure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It can also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety that is capable of or maintains at leasttwo functions. The functions can affect the same outcome or a differentoutcome. The structure that produces the function can be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention can encode a Citrin peptide (a first function) while thosenucleosides that comprise the encoding RNA are, in and of themselves,capable of extending the half-life of the RNA (second function). In thisexample, delivery of the bifunctional modified RNA to a subjectsuffering from a protein deficiency would produce not only a peptide orprotein molecule that can ameliorate or treat a disease or conditions,but would also maintain a population modified RNA present in the subjectfor a prolonged period of time. In other aspects, a bifunction modifiedmRNA can be a quimeric molecule comprising, for example, an RNA encodinga Citrin peptide (a first function) and a second protein either fused tofirst protein or co-expressed with the first protein.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide of the present invention can be considered biologicallyactive if even a portion of the polynucleotide is biologically active ormimics an activity considered biologically relevant.

Chimera: As used herein, “chimera” is an entity having two or moreincongruous or heterogeneous parts or regions. For example, a chimericmolecule can comprise a first part comprising a Citrin polypeptide, anda second part (e.g., genetically fused to the first part) comprising asecond therapeutic protein (e.g., a protein with a distinct enzymaticactivity, an antigen binding moiety, or a moiety capable of extendingthe plasma half life of Citrin, for example, an Fc region of anantibody).

Sequence Optimization: The term “sequence optimization” refers to aprocess or series of processes by which nucleobases in a referencenucleic acid sequence are replaced with alternative nucleobases,resulting in a nucleic acid sequence with improved properties, e.g.,improved protein expression or decreased immunogenicity.

In general, the goal in sequence optimization is to produce a synonymousnucleotide sequence than encodes the same polypeptide sequence encodedby the reference nucleotide sequence. Thus, there are no amino acidsubstitutions (as a result of codon optimization) in the polypeptideencoded by the codon optimized nucleotide sequence with respect to thepolypeptide encoded by the reference nucleotide sequence.

Codon substitution: The terms “codon substitution” or “codonreplacement” in the context of sequence optimization refer to replacinga codon present in a reference nucleic acid sequence with another codon.A codon can be substituted in a reference nucleic acid sequence, forexample, via chemical peptide synthesis or through recombinant methodsknown in the art. Accordingly, references to a “substitution” or“replacement” at a certain location in a nucleic acid sequence (e.g., anmRNA) or within a certain region or subsequence of a nucleic acidsequence (e.g., an mRNA) refer to the substitution of a codon at suchlocation or region with an alternative codon.

As used herein, the terms “coding region” and “region encoding” andgrammatical variants thereof, refer to an Open Reading Frame (ORF) in apolynucleotide that upon expression yields a polypeptide or protein.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers and isotopes of the structure depicted. As used herein,the term “stereoisomer” means any geometric isomer (e.g., cis- andtrans-isomer), enantiomer, or diastereomer of a compound. The presentdisclosure encompasses any and all stereoisomers of the compoundsdescribed herein, including stereomerically pure forms (e.g.,geometrically pure, enantiomerically pure, or diastereomerically pure)and enantiomeric and stereoisomeric mixtures, e.g., racemates.Enantiomeric and stereomeric mixtures of compounds and means ofresolving them into their component enantiomers or stereoisomers arewell-known. “Isotopes” refers to atoms having the same atomic number butdifferent mass numbers resulting from a different number of neutrons inthe nuclei. For example, isotopes of hydrogen include tritium anddeuterium. Further, a compound, salt, or complex of the presentdisclosure can be prepared in combination with solvent or watermolecules to form solvates and hydrates by routine methods.

Contacting: As used herein, the term “contacting” means establishing aphysical connection between two or more entities. For example,contacting a mammalian cell with a nanoparticle composition means thatthe mammalian cell and a nanoparticle are made to share a physicalconnection. Methods of contacting cells with external entities both invivo and ex vivo are well known in the biological arts. For example,contacting a nanoparticle composition and a mammalian cell disposedwithin a mammal can be performed by varied routes of administration(e.g., intravenous, intramuscular, intradermal, and subcutaneous) andcan involve varied amounts of nanoparticle compositions. Moreover, morethan one mammalian cell can be contacted by a nanoparticle composition.

Conservative amino acid substitution: A “conservative amino acidsubstitution” is one in which the amino acid residue in a proteinsequence is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, or histidine), acidic side chains (e.g., aspartic acid orglutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, or cysteine), nonpolar sidechains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, or tryptophan), beta-branched side chains(e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an aminoacid in a polypeptide is replaced with another amino acid from the sameside chain family, the amino acid substitution is considered to beconservative. In another aspect, a string of amino acids can beconservatively replaced with a structurally similar string that differsin order and/or composition of side chain family members.

Non-conservative amino acid substitution: Non-conservative amino acidsubstitutions include those in which (i) a residue having anelectropositive side chain (e.g., Arg, His or Lys) is substituted for,or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilicresidue (e.g., Ser or Thr) is substituted for, or by, a hydrophobicresidue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or prolineis substituted for, or by, any other residue, or (iv) a residue having abulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) issubstituted for, or by, one having a smaller side chain (e.g., Ala orSer) or no side chain (e.g., Gly).

Other amino acid substitutions can be readily identified by workers ofordinary skill. For example, for the amino acid alanine, a substitutioncan be taken from any one of D-alanine, glycine, beta-alanine,L-cysteine and D-cysteine. For lysine, a replacement can be any one ofD-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine,ornithine, or D-ornithine. Generally, substitutions in functionallyimportant regions that can be expected to induce changes in theproperties of isolated polypeptides are those in which (i) a polarresidue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, oralanine; (ii) a cysteine residue is substituted for (or by) any otherresidue; (iii) a residue having an electropositive side chain, e.g.,lysine, arginine or histidine, is substituted for (or by) a residuehaving an electronegative side chain, e.g., glutamic acid or asparticacid; or (iv) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having such a side chain, e.g.,glycine. The likelihood that one of the foregoing non-conservativesubstitutions can alter functional properties of the protein is alsocorrelated to the position of the substitution with respect tofunctionally important regions of the protein: some non-conservativesubstitutions can accordingly have little or no effect on biologicalproperties.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence can apply to the entire length of an polynucleotide orpolypeptide or can apply to a portion, region or feature thereof.

Controlled Release: As used herein, the term “controlled release” refersto a pharmaceutical composition or compound release profile thatconforms to a particular pattern of release to effect a therapeuticoutcome.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention can be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivering: As used herein, the term “delivering” means providing anentity to a destination. For example, delivering a polynucleotide to asubject can involve administering a nanoparticle composition includingthe polynucleotide to the subject (e.g., by an intravenous,intramuscular, intradermal, or subcutaneous route). Administration of ananoparticle composition to a mammal or mammalian cell can involvecontacting one or more cells with the nanoparticle composition.

Delivery Agent: As used herein, “delivery agent” refers to any substancethat facilitates, at least in part, the in vivo, in vitro, or ex vivodelivery of a polynucleotide to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Diastereomer: As used herein, the term “diastereomer,” meansstereoisomers that are not mirror images of one another and arenon-superimposable on one another.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Domain: As used herein, when referring to polypeptides, the term“domain” refers to a motif of a polypeptide having one or moreidentifiable structural or functional characteristics or properties(e.g., binding capacity, serving as a site for protein-proteininteractions).

Dosing regimen: As used herein, a “dosing regimen” or a “dosing regimen”is a schedule of administration or physician determined regimen oftreatment, prophylaxis, or palliative care.

Effective Amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that treats a protein deficiency(e.g., a Citrin deficiency), an effective amount of an agent is, forexample, an amount of mRNA expressing sufficient Citrin to ameliorate,reduce, eliminate, or prevent the signs and symptoms associated with theCitrin deficiency, as compared to the severity of the symptom observedwithout administration of the agent. The term “effective amount” can beused interchangeably with “effective dose,” “therapeutically effectiveamount,” or “therapeutically effective dose.”

Enantiomer: As used herein, the term “enantiomer” means each individualoptically active form of a compound of the invention, having an opticalpurity or enantiomeric excess (as determined by methods standard in theart) of at least 80% (i.e., at least 90% of one enantiomer and at most10% of the other enantiomer), at least 90%, or at least 98%.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround or encase.

Encapsulation Efficiency: As used herein, “encapsulation efficiency”refers to the amount of a polynucleotide that becomes part of ananoparticle composition, relative to the initial total amount ofpolynucleotide used in the preparation of a nanoparticle composition.For example, if 97 mg of polynucleotide are encapsulated in ananoparticle composition out of a total 100 mg of polynucleotideinitially provided to the composition, the encapsulation efficiency canbe given as 97%. As used herein, “encapsulation” can refer to complete,substantial, or partial enclosure, confinement, surrounding, orencasement.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence that encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Enhanced Delivery: As used herein, the term “enhanced delivery” meansdelivery of more (e.g., at least 1.5 fold more, at least 2-fold more, atleast 3-fold more, at least 4-fold more, at least 5-fold more, at least6-fold more, at least 7-fold more, at least 8-fold more, at least 9-foldmore, at least 10-fold more) of a polynucleotide by a nanoparticle to atarget tissue of interest (e.g., mammalian liver) compared to the levelof delivery of a polynucleotide by a control nanoparticle to a targettissue of interest (e.g., MC3, KC2, or DLinDMA). The level of deliveryof a nanoparticle to a particular tissue can be measured by comparingthe amount of protein produced in a tissue to the weight of said tissue,comparing the amount of polynucleotide in a tissue to the weight of saidtissue, comparing the amount of protein produced in a tissue to theamount of total protein in said tissue, or comparing the amount ofpolynucleotide in a tissue to the amount of total polynucleotide in saidtissue. It will be understood that the enhanced delivery of ananoparticle to a target tissue need not be determined in a subjectbeing treated, it can be determined in a surrogate such as an animalmodel (e.g., a rat model).

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an mRNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan mRNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an mRNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Ex Vivo: As used herein, the term “ex vivo” refers to events that occuroutside of an organism (e.g., animal, plant, or microbe or cell ortissue thereof). Ex vivo events can take place in an environmentminimally altered from a natural (e.g., in vivo) environment.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element. When referring to polypeptides,“features” are defined as distinct amino acid sequence-based componentsof a molecule. Features of the polypeptides encoded by thepolynucleotides of the present invention include surface manifestations,local conformational shape, folds, loops, half-loops, domains,half-domains, sites, termini or any combination thereof.

Formulation: As used herein, a “formulation” includes at least apolynucleotide and one or more of a carrier, an excipient, and adelivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins can comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells. In someembodiments, a fragment is a subsequences of a full length protein(e.g., Citrin) wherein N-terminal, and/or C-terminal, and/or internalsubsequences have been deleted. In some preferred aspects of the presentinvention, the fragments of a protein of the present invention arefunctional fragments.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized. Thus, a functional fragment of apolynucleotide of the present invention is a polynucleotide capable ofexpressing a functional Citrin fragment. As used herein, a functionalfragment of Citrin refers to a fragment of wild type Citrin (i.e., afragment of any of its naturally occurring isoforms), or a mutant orvariant thereof, wherein the fragment retains a least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% of thebiological activity of the corresponding full length protein.

Helper Lipid: As used herein, the term “helper lipid” refers to acompound or molecule that includes a lipidic moiety (for insertion intoa lipid layer, e.g., lipid bilayer) and a polar moiety (for interactionwith physiologic solution at the surface of the lipid layer). Typicallythe helper lipid is a phospholipid. A function of the helper lipid is to“complement” the amino lipid and increase the fusogenicity of thebilayer and/or to help facilitate endosomal escape, e.g., of nucleicacid delivered to cells. Helper lipids are also believed to be a keystructural component to the surface of the LNP.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Generally, the term “homology” implies anevolutionary relationship between two molecules. Thus, two moleculesthat are homologous will have a common evolutionary ancestor. In thecontext of the present invention, the term homology encompasses both toidentity and similarity.

In some embodiments, polymeric molecules are considered to be“homologous” to one another if at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers inthe molecule are identical (exactly the same monomer) or are similar(conservative substitutions). The term “homologous” necessarily refersto a comparison between at least two sequences (polynucleotide orpolypeptide sequences).

Identity: As used herein, the term “identity” refers to the overallmonomer conservation between polymeric molecules, e.g., betweenpolynucleotide molecules (e.g. DNA molecules and/or RNA molecules)and/or between polypeptide molecules. Calculation of the percentidentity of two polynucleotide sequences, for example, can be performedby aligning the two sequences for optimal comparison purposes (e.g.,gaps can be introduced in one or both of a first and a second nucleicacid sequences for optimal alignment and non-identical sequences can bedisregarded for comparison purposes). In certain embodiments, the lengthof a sequence aligned for comparison purposes is at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or 100% of the length of the reference sequence. Thenucleotides at corresponding nucleotide positions are then compared.When a position in the first sequence is occupied by the same nucleotideas the corresponding position in the second sequence, then the moleculesare identical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can beconsidered equivalent.

Suitable software programs are available from various sources, and foralignment of both protein and nucleotide sequences. One suitable programto determine percent sequence identity is bl2seq, part of the BLASTsuite of program available from the U.S. government's National Centerfor Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov).Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Othersuitable programs are, e.g., Needle, Stretcher, Water, or Matcher, partof the EMBOSS suite of bioinformatics programs and also available fromthe European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

Sequence alignments can be conducted using methods known in the art suchas MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.

Different regions within a single polynucleotide or polypeptide targetsequence that aligns with a polynucleotide or polypeptide referencesequence can each have their own percent sequence identity. It is notedthat the percent sequence identity value is rounded to the nearesttenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to80.2. It also is noted that the length value will always be an integer.

In certain aspects, the percentage identity “% ID” of a first amino acidsequence (or nucleic acid sequence) to a second amino acid sequence (ornucleic acid sequence) is calculated as % ID=100×(Y/Z), where Y is thenumber of amino acid residues (or nucleobases) scored as identicalmatches in the alignment of the first and second sequences (as alignedby visual inspection or a particular sequence alignment program) and Zis the total number of residues in the second sequence. If the length ofa first sequence is longer than the second sequence, the percentidentity of the first sequence to the second sequence will be higherthan the percent identity of the second sequence to the first sequence.

One skilled in the art will appreciate that the generation of a sequencealignment for the calculation of a percent sequence identity is notlimited to binary sequence-sequence comparisons exclusively driven byprimary sequence data. It will also be appreciated that sequencealignments can be generated by integrating sequence data with data fromheterogeneous sources such as structural data (e.g., crystallographicprotein structures), functional data (e.g., location of mutations), orphylogenetic data. A suitable program that integrates heterogeneous datato generate a multiple sequence alignment is T-Coffee, available atwww.tcoffee.org, and alternatively available, e.g., from the EBI. Itwill also be appreciated that the final alignment used to calculatepercent sequence identity can be curated either automatically ormanually.

Immune response: The term “immune response” refers to the action of, forexample, lymphocytes, antigen presenting cells, phagocytic cells,granulocytes, and soluble macromolecules produced by the above cells orthe liver (including antibodies, cytokines, and complement) that resultsin selective damage to, destruction of, or elimination from the humanbody of invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues. In some cases, theadministration of a nanoparticle comprising a lipid component and anencapsulated therapeutic agent can trigger an immune response, which canbe caused by (i) the encapsulated therapeutic agent (e.g., an mRNA),(ii) the expression product of such encapsulated therapeutic agent(e.g., a polypeptide encoded by the mRNA), (iii) the lipid component ofthe nanoparticle, or (iv) a combination thereof.

Inflammatory response: “Inflammatory response” refers to immuneresponses involving specific and non-specific defense systems. Aspecific defense system reaction is a specific immune system reaction toan antigen. Examples of specific defense system reactions includeantibody responses. A non-specific defense system reaction is aninflammatory response mediated by leukocytes generally incapable ofimmunological memory, e.g., macrophages, eosinophils and neutrophils. Insome aspects, an immune response includes the secretion of inflammatorycytokines, resulting in elevated inflammatory cytokine levels.

Inflammatory cytokines: The term “inflammatory cytokine” refers tocytokines that are elevated in an inflammatory response. Examples ofinflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine(C-X-C motif) ligand 1; also known as GROα, interferon-γ (IFNγ), tumornecrosis factor α (TNFα), interferon γ-induced protein 10 (IP-10), orgranulocyte-colony stimulating factor (G-CSF). The term inflammatorycytokines includes also other cytokines associated with inflammatoryresponses known in the art, e.g., interleukin-1 (IL-1), interleukin-8(IL-8), interleukin-12 (IL-12), interleukin-13 (Il-13), interferon α(IFN-α), etc.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Insertional and deletional variants: “Insertional variants” whenreferring to polypeptides are those with one or more amino acidsinserted immediately adjacent to an amino acid at a particular positionin a native or starting sequence. “Immediately adjacent” to an aminoacid means connected to either the alpha-carboxy or alpha-aminofunctional group of the amino acid. “Deletional variants” when referringto polypeptides are those with one or more amino acids in the native orstarting amino acid sequence removed. Ordinarily, deletional variantswill have one or more amino acids deleted in a particular region of themolecule.

Intact: As used herein, in the context of a polypeptide, the term“intact” means retaining an amino acid corresponding to the wild typeprotein, e.g., not mutating or substituting the wild type amino acid.Conversely, in the context of a nucleic acid, the term “intact” meansretaining a nucleobase corresponding to the wild type nucleic acid,e.g., not mutating or substituting the wild type nucleobase.

Ionizable amino lipid: The term “ionizable amino lipid” includes thoselipids having one, two, three, or more fatty acid or fatty alkyl chainsand a pH-titratable amino head group (e.g., an alkylamino ordialkylamino head group). An ionizable amino lipid is typicallyprotonated (i.e., positively charged) at a pH below the pKa of the aminohead group and is substantially not charged at a pH above the pKa. Suchionizable amino lipids include, but are not limited to DLin-MC3-DMA(MC3) and (13Z,165Z)—N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine(L608).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances (e.g., polynucleotides or polypeptides)can have varying levels of purity in reference to the substances fromwhich they have been isolated. Isolated substances and/or entities canbe separated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated substances are more than about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or more than about 99% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents.

Substantially isolated: By “substantially isolated” is meant that thecompound is substantially separated from the environment in which it wasformed or detected. Partial separation can include, for example, acomposition enriched in the compound of the present disclosure.Substantial separation can include compositions containing at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, or at leastabout 99% by weight of the compound of the present disclosure, or saltthereof.

A polynucleotide, vector, polypeptide, cell, or any compositiondisclosed herein which is “isolated” is a polynucleotide, vector,polypeptide, cell, or composition which is in a form not found innature. Isolated polynucleotides, vectors, polypeptides, or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some aspects, apolynucleotide, vector, polypeptide, or composition which is isolated issubstantially pure.

Isomer: As used herein, the term “isomer” means any tautomer,stereoisomer, enantiomer, or diastereomer of any compound of theinvention. It is recognized that the compounds of the invention can haveone or more chiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

Linker: As used herein, a “linker” refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker can be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form polynucleotide multimers (e.g.,through linkage of two or more chimeric polynucleotides molecules or IVTpolynucleotides) or polynucleotides conjugates, as well as to administera payload, as described herein. Examples of chemical groups that can beincorporated into the linker include, but are not limited to, alkyl,alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers andderivatives thereof, Other examples include, but are not limited to,cleavable moieties within the linker, such as, for example, a disulfidebond (—S—S—) or an azo bond (—N═N—), which can be cleaved using areducing agent or photolysis. Non-limiting examples of a selectivelycleavable bond include an amido bond can be cleaved for example by theuse of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,and/or photolysis, as well as an ester bond can be cleaved for exampleby acidic or basic hydrolysis.

Methods of Administration: As used herein, “methods of administration”can include intravenous, intramuscular, intradermal, subcutaneous, orother methods of delivering a composition to a subject. A method ofadministration can be selected to target delivery (e.g., to specificallydeliver) to a specific region or system of a body.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules can be modified inmany ways including chemically, structurally, and functionally. In someembodiments, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Nanoparticle Composition: As used herein, a “nanoparticle composition”is a composition comprising one or more lipids. Nanoparticlecompositions are typically sized on the order of micrometers or smallerand can include a lipid bilayer. Nanoparticle compositions encompasslipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), andlipoplexes. For example, a nanoparticle composition can be a liposomehaving a lipid bilayer with a diameter of 500 nm or less.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non-human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Nucleic acid sequence: The terms “nucleic acid sequence,” “nucleotidesequence,” or “polynucleotide sequence” are used interchangeably andrefer to a contiguous nucleic acid sequence. The sequence can be eithersingle stranded or double stranded DNA or RNA, e.g., an mRNA.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that comprises a polymer of nucleotides. These polymersare often referred to as polynucleotides. Exemplary nucleic acids orpolynucleotides of the invention include, but are not limited to,ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization), ethylene nucleicacids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids orcombinations thereof.

The phrase “nucleotide sequence encoding” refers to the nucleic acid(e.g., an mRNA or DNA molecule) coding sequence which encodes apolypeptide. The coding sequence can further include initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of an individual or mammal to which the nucleic acid isadministered. The coding sequence can further include sequences thatencode signal peptides.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g., alkyl) per se is optional.

Part: As used herein, a “part” or “region” of a polynucleotide isdefined as any portion of the polynucleotide that is less than theentire length of the polynucleotide.

Patient: As used herein, “patient” refers to a subject who can seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition. In some embodiments,the treatment is needed, required, or received to prevent or decreasethe risk of developing acute disease, i.e., it is a prophylactictreatment.

Citrin Associated Disease: As use herein the terms “Citrin-associateddisease” or “Citrin-associated disorder” refer to diseases or disorders,respectively, which result from aberrant Citrin activity (e.g.,decreased activity or increased activity). As a non-limiting example,Citrullinemia is a Citrin associated disease.

The terms “Citrin enzymatic activity” or “Citrin activity” are usedinterchangeably in the present disclosure and refer to Citrin's abilityto catalyze mitochondrial uptake of glutamate and/or export of aspartateand/or decrease ammonia. Accordingly, a fragment or variant retaining orhaving Citrin enzymatic activity or Citrin activity refers to a fragmentor variant that has measurable enzymatic catalyzing the uptake ofglutamate, export of aspartate or decrease of ammonia.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms that are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients can include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, acetic acid,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound that contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are used. Lists of suitable salts are foundin Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties,Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH,2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19(1977), each of which is incorporated herein by reference in itsentirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates can be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Polynucleotide: The term “polynucleotide” as used herein refers topolymers of nucleotides of any length, including ribonucleotides,deoxyribonucleotides, analogs thereof, or mixtures thereof. This termrefers to the primary structure of the molecule. Thus, the term includestriple-, double- and single-stranded deoxyribonucleic acid (“DNA”), aswell as triple-, double- and single-stranded ribonucleic acid (“RNA”).It also includes modified, for example by alkylation, and/or by capping,and unmodified forms of the polynucleotide. More particularly, the term“polynucleotide” includes polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), includingtRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, anyother type of polynucleotide which is an N- or C-glycoside of a purineor pyrimidine base, and other polymers containing normucleotidicbackbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”)and polymorpholino polymers, and other synthetic sequence-specificnucleic acid polymers providing that the polymers contain nucleobases ina configuration which allows for base pairing and base stacking, such asis found in DNA and RNA. In particular aspects, the polynucleotidecomprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. Insome aspects, the synthetic mRNA comprises at least one unnaturalnucleobase. In some aspects, all nucleobases of a certain class havebeen replaced with unnatural nucleobases (e.g., all uridines in apolynucleotide disclosed herein can be replaced with an unnaturalnucleobase, e.g., 5-methoxyuridine). In some aspects, the polynucleotide(e.g., a synthetic RNA or a synthetic DNA) comprises only naturalnucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T(thymidine) in the case of a synthetic DNA, or A, C, G, and U (uridine)in the case of a synthetic RNA.

The skilled artisan will appreciate that the T bases in the codon mapsdisclosed herein are present in DNA, whereas the T bases would bereplaced by U bases in corresponding RNAs. For example, acodon-nucleotide sequence disclosed herein in DNA form, e.g., a vectoror an in-vitro translation (IVT) template, would have its T basestranscribed as U based in its corresponding transcribed mRNA. In thisrespect, both codon-optimized DNA sequences (comprising T) and theircorresponding mRNA sequences (comprising U) are consideredcodon-optimized nucleotide sequence of the present invention. A skilledartisan would also understand that equivalent codon-maps can begenerated by replaced one or more bases with non-natural bases. Thus,e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map),which in turn would correspond to a ΨΨC codon (RNA map in which U hasbeen replaced with pseudouridine).

Standard A-T and G-C base pairs form under conditions which allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3and C4-NH2, of cytidine and the C2-NH2, N′—H and C6-oxy, respectively,of guanosine. Thus, for example, guanosine(2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-)results in a modified nucleotide which will not effectively base pairwith guanosine but will form a base pair with isoguanosine (U.S. Pat.No. 5,681,702 to Collins et al.). Isocytosine is available from SigmaChemical Co. (St. Louis, Mo.); isocytidine can be prepared by the methoddescribed by Switzer et al. (1993) Biochemistry 32:10489-10496 andreferences cited therein; 2′-deoxy-5-methyl-isocytidine can be preparedby the method of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 andreferences cited therein; and isoguanine nucleotides can be preparedusing the method described by Switzer et al., 1993, supra, and Mantschet al., 1993, Biochem. 14:5593-5601, or by the method described in U.S.Pat. No. 5,780,610 to Collins et al. Other nonnatural base pairs can besynthesized by the method described in Piccirilli et al., 1990, Nature343:33-37, for the synthesis of 2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modifiednucleotide units which form unique base pairs are known, such as thosedescribed in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 andSwitzer et al., supra.

Polypeptide: The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can comprise modified amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids such as homocysteine, ornithine, p-acetylphenylalanine,D-amino acids, and creatine), as well as other modifications known inthe art.

The term, as used herein, refers to proteins, polypeptides, and peptidesof any size, structure, or function. Polypeptides include encodedpolynucleotide products, naturally occurring polypeptides, syntheticpolypeptides, homologs, orthologs, paralogs, fragments and otherequivalents, variants, and analogs of the foregoing. A polypeptide canbe a monomer or can be a multi-molecular complex such as a dimer, trimeror tetramer. They can also comprise single chain or multichainpolypeptides. Most commonly disulfide linkages are found in multichainpolypeptides. The term polypeptide can also apply to amino acid polymersin which one or more amino acid residues are an artificial chemicalanalogue of a corresponding naturally occurring amino acid. In someembodiments, a “peptide” can be less than or equal to 50 amino acidslong, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acidslong.

Polypeptide variant: As used herein, the term “polypeptide variant”refers to molecules that differ in their amino acid sequence from anative or reference sequence. The amino acid sequence variants canpossess substitutions, deletions, and/or insertions at certain positionswithin the amino acid sequence, as compared to a native or referencesequence. Ordinarily, variants will possess at least about 50% identity,at least about 60% identity, at least about 70% identity, at least about80% identity, at least about 90% identity, at least about 95% identity,at least about 99% identity to a native or reference sequence. In someembodiments, they will be at least about 80%, or at least about 90%identical to a native or reference sequence.

Polypeptide per unit drug (PUD): As used herein, a PUD or product perunit drug, is defined as a subdivided portion of total daily dose,usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) asmeasured in body fluid or tissue, usually defined in concentration suchas pmol/mL, mmol/mL, etc. divided by the measure in the body fluid.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or more signs,symptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more signs, symptoms, features, ormanifestations of a particular infection, disease, disorder, and/orcondition; partially or completely delaying progression from aninfection, a particular disease, disorder and/or condition; and/ordecreasing the risk of developing pathology associated with theinfection, the disease, disorder, and/or condition.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Prophylactic: As used herein, “prophylactic” refers to a therapeutic orcourse of action used to prevent the spread of disease.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure takento maintain health and prevent the spread of disease. An “immuneprophylaxis” refers to a measure to produce active or passive immunityto prevent the spread of disease.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridine: As used herein, pseudouridine (ψ) refers to theC-glycoside isomer of the nucleoside uridine. A “pseudouridine analog”is any modification, variant, isoform or derivative of pseudouridine.For example, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methylpseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ), and2′-O-methyl-pseudouridine (ψm).

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Reference Nucleic Acid Sequence: The term “reference nucleic acidsequence” or “reference nucleic acid” or “reference nucleotide sequence”or “reference sequence” refers to a starting nucleic acid sequence(e.g., a RNA, e.g., an mRNA sequence) that can be sequence optimized. Insome embodiments, the reference nucleic acid sequence is a wild typenucleic acid sequence, a fragment or a variant thereof. In someembodiments, the reference nucleic acid sequence is a previouslysequence optimized nucleic acid sequence.

Salts: In some aspects, the pharmaceutical composition for deliverydisclosed herein and comprises salts of some of their lipidconstituents. The term “salt” includes any anionic and cationic complex.Non-limiting examples of anions include inorganic and organic anions,e.g., fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate),phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide,carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfate, sulfide,sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate,formate, acetate, benzoate, citrate, tartrate, lactate, acrylate,polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate,malate, mandelate, tiglate, ascorbate, salicylate, polymethacrylate,perchlorate, chlorate, chlorite, hypochlorite, bromate, hypobromite,iodate, an alkylsulfonate, an arylsulfonate, arsenate, arsenite,chromate, dichromate, cyanide, cyanate, thiocyanate, hydroxide,peroxide, permanganate, and mixtures thereof.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g., body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further can include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, or organs. A sample further refers to a medium,such as a nutrient broth or gel, which can contain cellular components,such as proteins or nucleic acid molecule.

Signal Sequence: As used herein, the phrases “signal sequence,” “signalpeptide,” and “transit peptide” are used interchangeably and refer to asequence that can direct the transport or localization of a protein to acertain organelle, cell compartment, or extracellular export. The termencompasses both the signal sequence polypeptide and the nucleic acidsequence encoding the signal sequence. Thus, references to a signalsequence in the context of a nucleic acid refer in fact to the nucleicacid sequence encoding the signal sequence polypeptide.

Signal transduction pathway: A “signal transduction pathway” refers tothe biochemical relationship between a variety of signal transductionmolecules that play a role in the transmission of a signal from oneportion of a cell to another portion of a cell. As used herein, thephrase “cell surface receptor” includes, for example, molecules andcomplexes of molecules capable of receiving a signal and thetransmission of such a signal across the plasma membrane of a cell.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Specific delivery: As used herein, the term “specific delivery,”“specifically deliver,” or “specifically delivering” means delivery ofmore (e.g., at least 1.5 fold more, at least 2-fold more, at least3-fold more, at least 4-fold more, at least 5-fold more, at least 6-foldmore, at least 7-fold more, at least 8-fold more, at least 9-fold more,at least 10-fold more) of a polynucleotide by a nanoparticle to a targettissue of interest (e.g., mammalian liver) compared to an off-targettissue (e.g., mammalian spleen). The level of delivery of a nanoparticleto a particular tissue can be measured by comparing the amount ofprotein produced in a tissue to the weight of said tissue, comparing theamount of polynucleotide in a tissue to the weight of said tissue,comparing the amount of protein produced in a tissue to the amount oftotal protein in said tissue, or comparing the amount of polynucleotidein a tissue to the amount of total polynucleotide in said tissue. Forexample, for renovascular targeting, a polynucleotide is specificallyprovided to a mammalian kidney as compared to the liver and spleen if1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold morepolynucleotide per 1 g of tissue is delivered to a kidney compared tothat delivered to the liver or spleen following systemic administrationof the polynucleotide. It will be understood that the ability of ananoparticle to specifically deliver to a target tissue need not bedetermined in a subject being treated, it can be determined in asurrogate such as an animal model (e.g., a rat model).

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and in some cases capable of formulation intoan efficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize,” “stabilized,”“stabilized region” means to make or become stable.

Stereoisomer: As used herein, the term “stereoisomer” refers to allpossible different isomeric as well as conformational forms that acompound can possess (e.g., a compound of any formula described herein),in particular all possible stereochemically and conformationallyisomeric forms, all diastereomers, enantiomers and/or conformers of thebasic molecular structure. Some compounds of the present invention canexist in different tautomeric forms, all of the latter being includedwithin the scope of the present invention.

Subject: By “subject” or “individual” or “animal” or “patient” or“mammal,” is meant any subject, particularly a mammalian subject, forwhom diagnosis, prognosis, or therapy is desired. Mammalian subjectsinclude, but are not limited to, humans, domestic animals, farm animals,zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs,rabbits, rats, mice, horses, cattle, cows; primates such as apes,monkeys, orangutans, and chimpanzees; canids such as dogs and wolves;felids such as cats, lions, and tigers; equids such as horses, donkeys,and zebras; bears, food animals such as cows, pigs, and sheep; ungulatessuch as deer and giraffes; rodents such as mice, rats, hamsters andguinea pigs; and so on. In certain embodiments, the mammal is a humansubject. In other embodiments, a subject is a human patient. In aparticular embodiment, a subject is a human patient in need oftreatment.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalcharacteristics rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemicalcharacteristics.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneous: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore signs and symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or can notexhibit signs and symptoms of the disease, disorder, and/or conditionbut harbors a propensity to develop a disease or its signs and symptoms.In some embodiments, an individual who is susceptible to a disease,disorder, and/or condition (for example, CTLN2) can be characterized byone or more of the following: (1) a genetic mutation associated withdevelopment of the disease, disorder, and/or condition; (2) a geneticpolymorphism associated with development of the disease, disorder,and/or condition; (3) increased and/or decreased expression and/oractivity of a protein and/or nucleic acid associated with the disease,disorder, and/or condition; (4) habits and/or lifestyles associated withdevelopment of the disease, disorder, and/or condition; (5) a familyhistory of the disease, disorder, and/or condition; and (6) exposure toand/or infection with a microbe associated with development of thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will developthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionwill not develop the disease, disorder, and/or condition.

Sustained release: As used herein, the term “sustained release” refersto a pharmaceutical composition or compound release profile thatconforms to a release rate over a specific period of time.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides or othermolecules of the present invention can be chemical or enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells can be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism can be ananimal, for example a mammal, a human, a subject or a patient.

Target tissue: As used herein “target tissue” refers to any one or moretissue types of interest in which the delivery of a polynucleotide wouldresult in a desired biological and/or pharmacological effect. Examplesof target tissues of interest include specific tissues, organs, andsystems or groups thereof. In particular applications, a target tissuecan be a kidney, a lung, a spleen orvascular endothelium in vessels(e.g., intra-coronary or intra-femoral). An “off-target tissue” refersto any one or more tissue types in which the expression of the encodedprotein does not result in a desired biological and/or pharmacologicaleffect.

The presence of a therapeutic agent in an off-target issue can be theresult of: (i) leakage of a polynucleotide from the administration siteto peripheral tissue or distant off-target tissue via diffusion orthrough the bloodstream (e.g., a polynucleotide intended to express apolypeptide in a certain tissue would reach the off-target tissue andthe polypeptide would be expressed in the off-target tissue); or (ii)leakage of an polypeptide after administration of a polynucleotideencoding such polypeptide to peripheral tissue or distant off-targettissue via diffusion or through the bloodstream (e.g., a polynucleotidewould expressed a polypeptide in the target tissue, and the polypeptidewould diffuse to peripheral tissue).

Targeting sequence: As used herein, the phrase “targeting sequence”refers to a sequence that can direct the transport or localization of aprotein or polypeptide.

Terminus: As used herein the terms “termini” or “terminus,” whenreferring to polypeptides, refers to an extremity of a peptide orpolypeptide. Such extremity is not limited only to the first or finalsite of the peptide or polypeptide but can include additional aminoacids in the terminal regions. The polypeptide based molecules of theinvention can be characterized as having both an N-terminus (terminatedby an amino acid with a free amino group (NH₂)) and a C-terminus(terminated by an amino acid with a free carboxyl group (COOH)).Proteins of the invention are in some cases made up of multiplepolypeptide chains brought together by disulfide bonds or bynon-covalent forces (multimers, oligomers). These sorts of proteins willhave multiple N- and C-termini. Alternatively, the termini of thepolypeptides can be modified such that they begin or end, as the casecan be, with a non-polypeptide based moiety such as an organicconjugate.

Therapeutic Agent: The term “therapeutic agent” refers to an agent that,when administered to a subject, has a therapeutic, diagnostic, and/orprophylactic effect and/or elicits a desired biological and/orpharmacological effect. For example, in some embodiments, an mRNAencoding a Citrin polypeptide can be a therapeutic agent.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve signs and symptoms of,diagnose, prevent, and/or delay the onset of the infection, disease,disorder, and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve signs and symptoms of,diagnose, prevent, and/or delay the onset of the infection, disease,disorder, and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr. period. The total daily dose can beadministered as a single unit dose or a split dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors can regulatetranscription of a target gene alone or in a complex with othermolecules.

Transcription: As used herein, the term “transcription” refers tomethods to produce mRNA (e.g., an mRNA sequence or template) from DNA(e.g., a DNA template or sequence).

Transfection: As used herein, “transfection” refers to the introductionof a polynucleotide (e.g., exogenous nucleic acids) into a cell whereina polypeptide encoded by the polynucleotide is expressed (e.g., mRNA) orthe polypeptide modulates a cellular function (e.g., siRNA, miRNA). Asused herein, “expression” of a nucleic acid sequence refers totranslation of a polynucleotide (e.g., an mRNA) into a polypeptide orprotein and/or post-translational modification of a polypeptide orprotein. Methods of transfection include, but are not limited to,chemical methods, physical treatments and cationic lipids or mixtures.

Treating, treatment, therapy: As used herein, the term “treating” or“treatment” or “therapy” refers to partially or completely alleviating,ameliorating, improving, relieving, delaying onset of, inhibitingprogression of, reducing severity of, and/or reducing incidence of oneor more signs and symptoms or features of a disease, e.g., Citrullinemia(e.g., CTLN2). For example, “treating” Citrullinemia (e.g., CTLN2) canrefer to diminishing signs and symptoms associated with the disease,prolong the lifespan (increase the survival rate) of patients, reducingthe severity of the disease, preventing or delaying the onset of thedisease, etc. Treatment can be administered to a subject who does notexhibit signs of a disease, disorder, and/or condition and/or to asubject who exhibits only early signs of a disease, disorder, and/orcondition for the purpose of decreasing the risk of developing pathologyassociated with the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in some way. Unmodified can,but does not always, refer to the wild type or native form of abiomolecule. Molecules can undergo a series of modifications wherebyeach modified molecule can serve as the “unmodified” starting moleculefor a subsequent modification.

Uracil: Uracil is one of the four nucleobases in the nucleic acid ofRNA, and it is represented by the letter U. Uracil can be attached to aribose ring, or more specifically, a ribofuranose via a β-N₁-glycosidicbond to yield the nucleoside uridine. The nucleoside uridine is alsocommonly abbreviated according to the one letter code of its nucleobase,i.e., U. Thus, in the context of the present disclosure, when a monomerin a polynucleotide sequence is U, such U is designated interchangeablyas a “uracil” or a “uridine.”

Uridine Content: The terms “uridine content” or “uracil content” areinterchangeable and refer to the amount of uracil or uridine present ina certain nucleic acid sequence. Uridine content or uracil content canbe expressed as an absolute value (total number of uridine or uracil inthe sequence) or relative (uridine or uracil percentage respect to thetotal number of nucleobases in the nucleic acid sequence).

Uridine Modified Sequence: The terms “uridine-modified sequence” refersto a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence)with a different overall or local uridine content (higher or loweruridine content) or with different uridine patterns (e.g., gradientdistribution or clustering) with respect to the uridine content and/oruridine patterns of a candidate nucleic acid sequence. In the content ofthe present disclosure, the terms “uridine-modified sequence” and“uracil-modified sequence” are considered equivalent andinterchangeable.

A “high uridine codon” is defined as a codon comprising two or threeuridines, a “low uridine codon” is defined as a codon comprising oneuridine, and a “no uridine codon” is a codon without any uridines. Insome embodiments, a uridine-modified sequence comprises substitutions ofhigh uridine codons with low uridine codons, substitutions of highuridine codons with no uridine codons, substitutions of low uridinecodons with high uridine codons, substitutions of low uridine codonswith no uridine codons, substitution of no uridine codons with lowuridine codons, substitutions of no uridine codons with high uridinecodons, and combinations thereof. In some embodiments, a high uridinecodon can be replaced with another high uridine codon. In someembodiments, a low uridine codon can be replaced with another lowuridine codon. In some embodiments, a no uridine codon can be replacedwith another no uridine codon. A uridine-modified sequence can beuridine enriched or uridine rarefied.

Uridine Enriched: As used herein, the terms “uridine enriched” andgrammatical variants refer to the increase in uridine content (expressedin absolute value or as a percentage value) in an sequence optimizednucleic acid (e.g., a synthetic mRNA sequence) with respect to theuridine content of the corresponding candidate nucleic acid sequence.Uridine enrichment can be implemented by substituting codons in thecandidate nucleic acid sequence with synonymous codons containing lessuridine nucleobases. Uridine enrichment can be global (i.e., relative tothe entire length of a candidate nucleic acid sequence) or local (i.e.,relative to a subsequence or region of a candidate nucleic acidsequence).

Uridine Rarefied: As used herein, the terms “uridine rarefied” andgrammatical variants refer to a decrease in uridine content (expressedin absolute value or as a percentage value) in an sequence optimizednucleic acid (e.g., a synthetic mRNA sequence) with respect to theuridine content of the corresponding candidate nucleic acid sequence.Uridine rarefication can be implemented by substituting codons in thecandidate nucleic acid sequence with synonymous codons containing lessuridine nucleobases. Uridine rarefication can be global (i.e., relativeto the entire length of a candidate nucleic acid sequence) or local(i.e., relative to a subsequence or region of a candidate nucleic acidsequence).

Variant: The term variant as used in present disclosure refers to bothnatural variants (e.g., polymorphisms, isoforms, etc.) and artificialvariants in which at least one amino acid residue in a native orstarting sequence (e.g., a wild type sequence) has been removed and adifferent amino acid inserted in its place at the same position. Thesevariants can de described as “substitutional variants.” Thesubstitutions can be single, where only one amino acid in the moleculehas been substituted, or they can be multiple, where two or more aminoacids have been substituted in the same molecule. If amino acids areinserted or deleted, the resulting variant would be an “insertionalvariant” or a “deletional variant” respectively.

30. Embodiments

Throughout this section, the term embodiment is abbreviated as ‘E’followed by an ordinal. For example, E1 is equivalent to Embodiment 1.

E1. A polynucleotide comprising an open reading frame (ORF) encoding aCitrin polypeptide, wherein the uracil or thymine content of the ORFrelative to the theoretical minimum uracil or thymine content of anucleotide sequence encoding the Citrin polypeptide (% UTM or % TTM), isbetween about 100% and about 150%.

E2. The polynucleotide of E1, wherein the % UTM or % TTM is betweenabout 105% and about 145%, about 105% and about 140%, about 110% andabout 140%, about 110% and about 145%, about 115% and about 135%, about105% and about 135%, about 110% and about 135%, about 115% and about145%, about 115% and about 140%, about 115% and about 135%, about 115%and about 130%, or about 117% and about 126%.

E3. The polynucleotide of E2, wherein the % UTM or % TTM is between (i)110%, 111%, 112%, 113%, 114%, 115%, 116%, or 117%, and (ii) 126%, 127%,128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%,or 140%.

E4. The polynucleotide of any one of E1 to E3, wherein the uracil orthymine content of the ORF relative to the uracil or thymine content ofthe corresponding wild-type ORF (% UWT or % TWT) is less than 100%.

E5. The polynucleotide of E4, wherein the % UWT or % TWT is less thanabout 95%, less than about 90%, less than about 85%, less than about80%, less than about 75%, less than about 70%, less than 69%, less than68%, less than 67%, less than 66%, or less than 65%.

E6. The polynucleotide of E4, wherein the % UWT or % TWT is between 60%and 65%.

E7. The polynucleotide of any one of E1 to E6, wherein the uracil orthymine content in the ORF relative to the total nucleotide content inthe ORF (% UTL or % TTL) is less than about 50%, less than about 40%,less than about 30%, or less than about 20%.

E8. The polynucleotide of E7, wherein the % UTL or % TTL is less thanabout 18%.

E9. The polynucleotide of any one of E1 to E8, wherein the % UTL or %TTL is between about 15% and about 18%.

E10. The polynucleotide of any one of E1 to E9, wherein the guaninecontent of the ORF with respect to the theoretical maximum guaninecontent of a nucleotide sequence encoding the Citrin polypeptide (%GTMX) is at least 69%, at least 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or about100%.

E11. The polynucleotide of E10, wherein the % GTMX is between about 68%and about 80%, between about 70% and about 80%, between about 70% andabout 79%, between about 70% and about 78%, between about 70% and about77%, or between about 70% and about 76%.

E12. The polynucleotide of any one of E1 to E11, wherein the cytosinecontent of the ORF relative to the theoretical maximum cytosine contentof a nucleotide sequence encoding the Citrin polypeptide (% CTMX) is atleast 60%, at least 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or about 100%.

E13. The polynucleotide of E12, wherein the % CTMX is between about 60%and about 80%, between about 65% and about 80%, between about 65% andabout 75%, or between about 69% and about 75%.

E14. The polynucleotide of any one of E1 to E13, wherein the guanine andcytosine content (G/C) of the ORF relative to the theoretical maximumG/C content of a nucleotide sequence encoding the Citrin polypeptide (%G/CTMX) is at least about 81%, at least about 85%, at least about 90%,at least about 95%, or about 100%.

E15. The polynucleotide of any one of E1 to E13, wherein the % G/CTMX isbetween about 80% and about 100%, between about 80% and about 95%,between about 85% and about 95%, between about 90% and about 95%,between about 91% and about 95%, or between about 91% and about 94%.

E16. The polynucleotide of any one of E1 to E15, wherein the G/C contentin the ORF relative to the G/C content in the corresponding wild-typeORF (% G/CWT) is at least 102%, at least 103%, at least 104%, at least105%, at least 106%, at least 107%, at least 110%, at least 115%, atleast 120%, at least 125%, at least 130%, at least 131%, at least 132%,at least 133%, or at least 134%.

E17. The polynucleotide of any one of E1 to E15, wherein the average G/Ccontent in the 3rd codon position in the ORF is at least 30%, at least35%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, or at least49% higher than the average G/C content in the 3rd codon position in thecorresponding wild-type ORF.

E18. The polynucleotide of any one of E1 to E17, wherein the ORF furthercomprises at least one low-frequency codon.

E19. The polynucleotide of any one of E1 to E18,

-   -   (i) wherein the ORF is at least 79%, at least 80%, at least 81%,        at least 82%, at least 83%, at least 84%, at least 85%, at least        86%, at least 87%, at least 88%, at least 89%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%, or        100% identical to CITRIN-CO6, CITRIN-CO12, CITRIN-CO23,        CITRIN-CO11, or CITRIN-CO25,    -   (ii) wherein the ORF is at least 78%, at least 79%, at least        80%, at least 81%, at least 82%, at least 83%, at least 84%, at        least 85%, at least 86%, at least 87%, at least 88%, at least        89%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99%, or 100% identical to CITRIN-CO2, CITRIN-CO16,        CITRIN-CO20, CITRIN-CO17, CITRIN-CO4, or CITRIN-CO1,    -   (iii) wherein the ORF is at least 76%, at least 77%, at least        78%, at least 79%, at least 80%, at least 81%, at least 82%, at        least 83%, at least 84%, at least 85%, at least 86%, at least        87%, at least 88%, at least 89%, at least 90%, at least 91%, at        least 92%, at least 93%, at least 94%, at least 95%, at least        96%, at least 97%, at least 98%, at least 99%, or 100% identical        to CITRIN-CO9, CITRIN-CO18, CITRIN-CO5, CITRIN-CO10,        CITRIN-CO13, or CITRIN-CO7,    -   (iv) wherein the ORF is at least 75%, at least 76%, at least        77%, at least 78%, at least 79%, at least 80%, at least 81%, at        least 82%, at least 83%, at least 84%, at least 85%, at least        86%, at least 87%, at least 88%, at least 89%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%, or        100% identical to CITRIN-CO15, CITRIN-CO14, CITRIN-CO3,        CITRIN-CO22, or CITRIN-CO19, or    -   (v) wherein the ORF is at least 74%, at least 75%, at least 76%,        at least 77%, at least 78%, at least 79%, at least 80%, at least        81%, at least 82%, at least 83%, at least 84%, at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99%, or 100% identical to CITRIN-CO24, CITRIN-CO8,        or CITRIN-CO21.

E20. A polynucleotide comprising an ORF,

-   -   (i) wherein the ORF is at least 79%, at least 80%, at least 81%,        at least 82%, at least 83%, at least 84%, at least 85%, at least        86%, at least 87%, at least 88%, at least 89%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%, or        100% identical to CITRIN-CO6, CITRIN-CO12, CITRIN-CO23,        CITRIN-CO11, or CITRIN-CO25,    -   (ii) wherein the ORF is at least 78%, at least 79%, at least        80%, at least 81%, at least 82%, at least 83%, at least 84%, at        least 85%, at least 86%, at least 87%, at least 88%, at least        89%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99%, or 100% identical to CITRIN-CO2, CITRIN-CO16,        CITRIN-CO20, CITRIN-CO17, CITRIN-CO4, or CITRIN-CO1,    -   (iii) wherein the ORF is at least 76%, at least 77%, at least        78%, at least 79%, at least 80%, at least 81%, at least 82%, at        least 83%, at least 84%, at least 85%, at least 86%, at least        87%, at least 88%, at least 89%, at least 90%, at least 91%, at        least 92%, at least 93%, at least 94%, at least 95%, at least        96%, at least 97%, at least 98%, at least 99%, or 100% identical        to CITRIN-CO9, CITRIN-CO18, CITRIN-CO5, CITRIN-CO10,        CITRIN-CO13, or CITRIN-CO7,    -   (iv) wherein the ORF is at least 75%, at least 76%, at least        77%, at least 78%, at least 79%, at least 80%, at least 81%, at        least 82%, at least 83%, at least 84%, at least 85%, at least        86%, at least 87%, at least 88%, at least 89%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99%, or        100% identical to CITRIN-CO15, CITRIN-CO14, CITRIN-CO3,        CITRIN-CO22, or CITRIN-CO19, or    -   (v) wherein the ORF is at least 74%, at least 75%, at least 76%,        at least 77%, at least 78%, at least 79%, at least 80%, at least        81%, at least 82%, at least 83%, at least 84%, at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99%, or 100% identical to CITRIN-CO24, CITRIN-CO8,        or CITRIN-CO21.

E21. The polynucleotide of any one of E1 to E20, wherein the ORF has atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NOs: 5-29.

E22. The polynucleotide of any one of E1 to E21, wherein the Citrinpolypeptide comprises an amino acid sequence at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or about 100% identical to (i) the polypeptide sequence of wildtype Citrin, isoform 1 (SEQ ID NO: 1), or (ii) the polypeptide sequenceof wild type Citrin, isoform 2 (SEQ ID NO: 3), and wherein the Citrinpolypeptide has aspartate or glutamate transport activity.

E23. The polynucleotide of E22, wherein the Citrin polypeptide is avariant, derivative, or mutant having aspartate or glutamate transportactivity.

E24. The polynucleotide of any one of E1 to E23, wherein thepolynucleotide sequence further comprises a nucleotide sequence encodinga transit peptide.

E25. The polynucleotide of any one of E1 to E24, wherein thepolynucleotide is single stranded.

E26. The polynucleotide of any one of E1 to E24, wherein thepolynucleotide is double stranded.

E27. The polynucleotide of any one of E1 to E26, wherein thepolynucleotide is DNA.

E28. The polynucleotide of any one of E1 to E26, wherein thepolynucleotide is RNA.

E29. The polynucleotide of E28, wherein the polynucleotide is mRNA.

E30. The polynucleotide of any one of E1 to E29, wherein thepolynucleotide comprises at least one chemically modified nucleobase,sugar, backbone, or any combination thereof.

E31. The polynucleotide of E30, wherein the at least one chemicallymodified nucleobase is selected from the group consisting ofpseudouracil (ψ), N1-methylpseudouracil (m1ψ), 2-thiouracil (s2U),4′-thiouracil, 5-methylcytosine, 5-methyluracil, and any combinationthereof.

E32. The polynucleotide of E30, wherein the at least one chemicallymodified nucleobase is 5-methoxyuracil.

E33. The polynucleotide of E32, wherein at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 99%, or 100% of the uracils are5-methoxyuracils.

E34. The polynucleotide of any one of E1 to E33, wherein thepolynucleotide further comprises a miRNA binding site.

E35. The polynucleotide of E34, wherein the miRNA binding site comprisesone or more nucleotide sequences selected from Table 3.

E36. The polynucleotide of E34, wherein the miRNA binding site binds tomiR-142.

E37. The polynucleotide of E35 or E36, wherein the miRNA binding sitebinds to miR-142-3p or miR-142-5p.

E38. The polynucleotide of E36 or E37, wherein the miR142 comprises SEQID NO: 73.

E39. The polynucleotide of any one of E1 to E38, wherein thepolynucleotide further comprises a 5′ UTR.

E40. The polynucleotide of E39, wherein the 5′ UTR comprises a nucleicacid sequence at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, orabout 100% identical to a sequence listed in SEQ ID NOs: 30-47.

E41. The polynucleotide of any one of E1 to E40, wherein thepolynucleotide further comprises a 3′ UTR.

E42. The polynucleotide of E41, wherein the 3′ UTR comprises a nucleicacid sequence at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, orabout 100% identical to a sequence listed in SEQ ID NOs: 48-72.

E43. The polynucleotide of E41 or E42, wherein the miRNA binding site islocated within the 3′ UTR.

E44. The polynucleotide of any one of E1 to E43, wherein thepolynucleotide further comprises a 5′ terminal cap.

E45. The polynucleotide of E44, wherein the 5′ terminal cap comprises aCap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof.

E46. The polynucleotide of any one of E1 to E45, wherein thepolynucleotide further comprises a poly-A region.

E47. The polynucleotide of E46, wherein the poly-A region is at leastabout 10, at least about 20, at least about 30, at least about 40, atleast about 50, at least about 60, at least about 70, at least about 80,or at least about 90 nucleotides in length.

E48. The polynucleotide of E47, wherein the poly-A region has about 10to about 200, about 20 to about 180, about 50 to about 160, about 70 toabout 140, or about 80 to about 120 nucleotides in length.

E49. The polynucleotide of any one of E1 to E48, wherein thepolynucleotide encodes a Citrin polypeptide that is fused to one or moreheterologous polypeptides.

E50. The polynucleotide of E49, wherein the one or more heterologouspolypeptides increase a pharmacokinetic property of the Citrinpolypeptide.

E51. The polynucleotide of any one of E1 to E50, wherein uponadministration to a subject, the polynucleotide has:

-   -   (i) a longer plasma half-life;    -   (ii) increased expression of a Citrin polypeptide encoded by the        ORF;    -   (iii) a lower frequency of arrested translation resulting in an        expression fragment;    -   (iv) greater structural stability; or    -   (v) any combination thereof,    -   relative to a corresponding polynucleotide comprising SEQ ID NO:        2 or 4.

E52. The polynucleotide of any one of E1 to E51, wherein thepolynucleotide comprises:

-   -   (i) a 5′-terminal cap;    -   (ii) a 5′-UTR;    -   (iii) an ORF encoding a Citrin polypeptide;    -   (iv) a 3′-UTR; and    -   (v) a poly-A region.

E53. The polynucleotide of E52, wherein the 3′-UTR comprises a miRNAbinding site.

E54. A method of producing the polynucleotide of any one of E1 to E53,the method comprising modifying an ORF encoding a Citrin polypeptide bysubstituting at least one uracil nucleobase with an adenine, guanine, orcytosine nucleobase, or by substituting at least one adenine, guanine,or cytosine nucleobase with a uracil nucleobase, wherein all thesubstitutions are synonymous substitutions.

E55. The method of E54, wherein the method further comprises replacingat least about 90%, at least about 95%, at least about 99%, or about100% of uracils with 5-methoxyuracils.

E56. A composition comprising

-   -   (a) the polynucleotide of any one of E1 to E53; and    -   (b) a delivery agent.

E57. The composition of E56, wherein the delivery agent comprises alipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymericcompound, a peptide, a protein, a cell, a nanoparticle mimic, ananotube, or a conjugate.

E58. The composition of E56, wherein the delivery agent comprises alipid nanoparticle.

E59. The composition of E58, wherein the lipid nanoparticle comprises alipid selected from the group consisting of DLin-DMA, DLin-K-DMA,98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG,PEGylated lipids, amino alcohol lipids, KL22, and any combinationsthereof.

E60. The composition of any one of E56 to E59, wherein the deliveryagent comprises a compound having the formula (I)

or a salt or stereoisomer thereof, wherein

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —CH₂)_(n)Q, —CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n        is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and    -   provided when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or        —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5,        or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is        1 or 2.

E61. A composition comprising a nucleotide sequence encoding a Citrinpolypeptide and a delivery agent, wherein the delivery agent comprises acompound having the formula (I)

or a salt or stereoisomer thereof, wherein

-   -   R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n        is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and a heteroaryl        group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H; each R is independently selected from the group        consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl; C3-14 alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and    -   provided when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or        —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5,        or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is        1 or 2.

E62. The composition of E60 or E61, wherein the compound is of Formula(IA):

or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5;    -   m is selected from 5, 6, 7, 8, and 9;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 1,        2, 3, 4, or 5 and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl        group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

E63. The composition of any one of E60 to E62, wherein m is 5, 7, or 9.

E64. The composition of any one of E60 to E63, wherein the compound isof Formula (II):

or a salt or stereoisomer thereof, wherein

-   -   l is selected from 1, 2, 3, 4, and 5;    -   M₁ is a bond or M′;    -   R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2,        3, or 4 and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl        group; and    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

E65. The composition of any one of E62 to E64, wherein M1 is M′.

E66. The composition of E65, wherein M and M′ are independently —C(O)O—or —OC(O)—.

E67. The composition of any one of E62 to E66, wherein 1 is 1, 3, or 5.

E68. The composition of E60 or E61, wherein the compound is selectedfrom the group consisting of Compound 1 to Compound 147, salts andstereoisomers thereof, and any combination thereof.

E69. The composition of E60 or E61, wherein the compound is of theFormula (IIa),

or a salt or stereoisomer thereof.

E70. The composition of E60 or E61, wherein the compound is of theFormula (IIb),

or a salt or stereoisomer thereof.

E71. The composition of E60 or E61, wherein the compound is of theFormula (IIc) or (IIe),

or a salt or stereoisomer thereof.

E72. The composition of any one of E69 to E71, wherein R₄ is selectedfrom —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.

E73. The composition of E60 or E61, wherein the compound is of theFormula (IId),

or a salt or stereoisomer thereof,

-   -   wherein R₂ and R₃ are independently selected from the group        consisting of C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl, n is selected from        2, 3, and 4, and R′, R″, R₅, R₆ and m are as defined in E60 or        E61.

E74. The composition of E73, wherein R₂ is C₈ alkyl.

E75. The composition of E74, wherein R₃ is C₅ alkyl, C₆ alkyl, C₇ alkyl,C₈ alkyl, or C₉ alkyl.

E76. The composition of any one of E73 to E75, wherein m is 5, 7, or 9.

E77. The composition of any one of E73 to E76, wherein each R₅ is H.

E78. The composition of E77, wherein each R₆ is H.

E79. The composition of any one of E60 to E78, which is a nanoparticlecomposition.

E80. The composition of E79, wherein the delivery agent furthercomprises a phospholipid.

E81. The composition of E80, wherein the phospholipid is selected fromthe group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine(DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and any mixtures thereof.

E82. The composition of any one of E60 to E81, wherein the deliveryagent further comprises a structural lipid.

E83. The composition of E82, wherein the structural lipid is selectedfrom the group consisting of cholesterol, fecosterol, sitosterol,ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine,ursolic acid, alpha-tocopherol, and any mixtures thereof.

E84. The composition of any one of E60 to E83, wherein the deliveryagent further comprises a PEG lipid.

E85. The composition of E84, wherein the PEG lipid is selected from thegroup consisting of a PEG-modified phosphatidylethanolamine, aPEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modifieddialkylamine, a PEG-modified diacylglycerol, a PEG-modifieddialkylglycerol, and any mixtures thereof.

E86. The composition of any one of E60 to E85, wherein the deliveryagent further comprises an ionizable lipid selected from the groupconsisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).

E87. The composition of any one of E60 to E86, wherein the deliveryagent further comprises a phospholipid, a structural lipid, a PEG lipid,or any combination thereof.

E88. The composition of any one of E60 to E87, wherein the compositionis formulated for in vivo delivery.

E89. The composition according any one of E60 to E88, which isformulated for intramuscular, subcutaneous, or intradermal delivery.

E90. A host cell comprising the polynucleotide of any one of E1 to E53.

E91. The host cell of E90, wherein the host cell is a eukaryotic cell.

E92. A vector comprising the polynucleotide of any one of E1 to E53.

E93. A method of making a polynucleotide comprising enzymatically orchemically synthesizing the polynucleotide of any one of E1 to E53.

E94. A polypeptide encoded by the polynucleotide of any one of E1 toE53, the composition of any one of E56 to E89, the host cell of E90 orE91, or the vector of E92 or produced by the method of E93.

E95. A method of expressing in vivo an active Citrin polypeptide in asubject in need thereof comprising administering to the subject aneffective amount of the polynucleotide of any one of E1 to E53, thecomposition of any one of E56 to E89, the host cell of E90 or E91, orthe vector of E92.

E96. A method of treating citrullinemia type 2 (CTLN2) in a subject inneed thereof comprising administering to the subject a therapeuticallyeffective amount of the polynucleotide of any one of E1 to E53, thecomposition of any one of E56 to E89, the host cell of E90 or E91, orthe vector of E92, wherein the administration alleviates the signs orsymptoms of CTLN2 in the subject.

E97. A method to prevent or delay the onset of CTLN2 signs or symptomsin a subject in need thereof comprising administering to the subject aprophylactically effective amount of the polynucleotide of any one of E1to E53, the composition of any one of E56 to E89, the host cell of E90or E91, or the vector of E92 before CTLN2 signs or symptoms manifest,wherein the administration prevents or delays the onset of CTLN2 signsor symptoms in the subject.

E98. A method to ameliorate the signs or symptoms of CTLN2 in a subjectin need thereof comprising administering to the subject atherapeutically effective amount of the polynucleotide of any one of E1to E53, the composition of any one of E56 to E89, the host cell of E90or E91, or the vector of E92 before CTLN2 signs or symptoms manifest,wherein the administration ameliorates CTLN2 signs or symptoms in thesubject.

31. Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” can mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art can be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they can beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1

Chimeric Polynucleotide Synthesis

A. Triphosphate Route

Two regions or parts of a chimeric polynucleotide can be joined orligated using triphosphate chemistry. According to this method, a firstregion or part of 100 nucleotides or less can be chemically synthesizedwith a 5′ monophosphate and terminal 3′desOH or blocked OH. If theregion is longer than 80 nucleotides, it can be synthesized as twostrands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus canfollow. Monophosphate protecting groups can be selected from any ofthose known in the art.

The second region or part of the chimeric polynucleotide can besynthesized using either chemical synthesis or IVT methods. IVT methodscan include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 80 nucleotides can be chemicallysynthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase,followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then such region or part can comprise a phosphate-sugarbackbone.

Ligation can then be performed using any known click chemistry,orthoclick chemistry, solulink, or other bioconjugate chemistries knownto those in the art.

B. Synthetic route

The chimeric polynucleotide can be made using a series of startingsegments. Such segments include:

-   -   (a) Capped and protected 5′ segment comprising a normal 3′OH        (SEG. 1)    -   (b) 5′ triphosphate segment which can include the coding region        of a polypeptide and comprising a normal 3′OH (SEG. 2)    -   (c) 5′ monophosphate segment for the 3′ end of the chimeric        polynucleotide (e.g., the tail) comprising cordycepin or no 3′OH        (SEG. 3)    -   After synthesis (chemical or IVT), segment 3 (SEG. 3) can be        treated with cordycepin and then with pyrophosphatase to create        the 5′monophosphate.

Segment 2 (SEG. 2) can then be ligated to SEG. 3 using RNA ligase. Theligated polynucleotide can then be purified and treated withpyrophosphatase to cleave the diphosphate. The treated SEG.2-SEG. 3construct is then purified and SEG. 1 is ligated to the 5′ terminus. Afurther purification step of the chimeric polynucleotide can beperformed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments can be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step can be as much as 90-95%.

Example 2 PCR for cDNA Production

PCR procedures for the preparation of cDNA can be performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 μM) 0.75 μl; ReversePrimer (10 μM) 0.75 μl; Template cDNA −100 ng; and dH₂O diluted to 25.0μl. The PCR reaction conditions can be: at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention can incorporate a poly-T₁₂₀for a poly-A₁₂₀ in the mRNA. Other reverse primers with longer orshorter poly(T) tracts can be used to adjust the length of the poly(A)tail in the polynucleotide mRNA.

The reaction can be cleaned up using Invitrogen's PURELINK™ PCR MicroKit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg).Larger reactions will require a cleanup using a product with a largercapacity. Following the cleanup, the cDNA can be quantified using theNANODROP™ and analyzed by agarose gel electrophoresis to confirm thecDNA is the expected size. The cDNA can then be submitted for sequencinganalysis before proceeding to the in vitro transcription reaction.

Example 3 In Vitro Transcription (IVT)

The in vitro transcription reactions can generate polynucleotidescontaining uniformly modified polynucleotides. Such uniformly modifiedpolynucleotides can comprise a region or part of the polynucleotides ofthe invention. The input nucleotide triphosphate (NTP) mix can be madeusing natural and un-natural NTPs.

A typical in vitro transcription reaction can include the following:

-   1 Template cDNA—1.0 μh-   2 10× transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl₂, 50    mM DTT, 10 mM Spermidine)—2.0 μl-   3 Custom NTPs (25 mM each)—7.2 μl-   4 RNase Inhibitor—20 U-   5 T7 RNA polymerase—3000 U-   6 dH₂O—Up to 20.0 μl, and-   7 Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix can be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase can then be used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA can bepurified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA can be quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4 Enzymatic Capping

Capping of a polynucleotide can be performed with a mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture can be incubatedat 65° C. for 5 minutes to denature RNA, and then can be transferredimmediately to ice.

The protocol can then involve the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The polynucleotide can then be purified using Ambion's MEGACLEAR™ Kit(Austin, Tex.) following the manufacturer's instructions. Following thecleanup, the RNA can be quantified using the NANODROP™ (ThermoFisher,Waltham, Mass.) and analyzed by agarose gel electrophoresis to confirmthe RNA is the proper size and that no degradation of the RNA hasoccurred. The RNA product can also be sequenced by running areverse-transcription-PCR to generate the cDNA for sequencing.

Example 5 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This can be done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂) (12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubating at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction can be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis, in some cases, a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction does not always result in an exact size polyA tail.Hence polyA tails of approximately between 40-200 nucleotides, e.g.,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe invention.

Example 6 Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides can be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA can becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure can be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure can be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure can be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes can be derived from a recombinant source.

When transfected into mammalian cells, the modified mRNAs can have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7 Capping Assays A. Protein Expression Assay

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be transfected into cells at equal concentrations.After 6, 12, 24 and 36 hours post-transfection, the amount of proteinsecreted into the culture medium can be assayed by ELISA. Syntheticpolynucleotides that secrete higher levels of protein into the mediumwould correspond to a synthetic polynucleotide with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be compared for purity using denaturing Agarose-Ureagel electrophoresis or HPLC analysis. Polynucleotides with a single,consolidated band by electrophoresis correspond to the higher purityproduct compared to polynucleotides with multiple bands or streakingbands. Synthetic polynucleotides with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure polynucleotide population.

C. Cytokine Analysis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be transfected into cells at multiple concentrations.After 6, 12, 24 and 36 hours post-transfection the amount ofpro-inflammatory cytokines such as TNF-alpha and IFN-beta secreted intothe culture medium can be assayed by ELISA. Polynucleotides resulting inthe secretion of higher levels of pro-inflammatory cytokines into themedium would correspond to polynucleotides containing animmune-activating cap structure.

D. Capping Reaction Efficiency

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be analyzed for capping reaction efficiency by LC-MSafter nuclease treatment. Nuclease treatment of capped polynucleotideswould yield a mixture of free nucleotides and the capped5′-5-triphosphate cap structure detectable by LC-MS. The amount ofcapped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and would correspond to cappingreaction efficiency. The cap structure with higher capping reactionefficiency would have a higher amount of capped product by LC-MS.

Example 8 Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) can be loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9 Nanodrop Modified RNA Quantification and UV Spectral Data

Modified polynucleotides in TE buffer (1 μl) can be used for Nanodrop UVabsorbance readings to quantitate the yield of each polynucleotide froman chemical synthesis or in vitro transcription reaction.

Example 10 Formulation of Modified mRNA Using Lipidoids

Polynucleotides can be formulated for in vitro experiments by mixing thepolynucleotides with the lipidoid at a set ratio prior to addition tocells. In vivo formulation can require the addition of extra ingredientsto facilitate circulation throughout the body. To test the ability ofthese lipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations can be used asa starting point. After formation of the particle, polynucleotide can beadded and allowed to integrate with the complex. The encapsulationefficiency can be determined using a standard dye exclusion assays.

Example 11 Method of Screening for Protein Expression A. ElectrosprayIonization

A biological sample that can contain proteins encoded by apolynucleotide administered to the subject can be prepared and analyzedaccording to the manufacturer protocol for electrospray ionization (ESI)using 1, 2, 3 or 4 mass analyzers. A biologic sample can also beanalyzed using a tandem ESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, can be compared toknown controls for a given protein and identity can be determined bycomparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample that can contain proteins encoded by one or morepolynucleotides administered to the subject can be prepared and analyzedaccording to the manufacturer protocol for matrix-assisted laserdesorption/ionization (MALDI).

Patterns of protein fragments, or whole proteins, can be compared toknown controls for a given protein and identity can be determined bycomparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which can contain proteins encoded by one or morepolynucleotides, can be treated with a trypsin enzyme to digest theproteins contained within. The resulting peptides can be analyzed byliquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS).The peptides can be fragmented in the mass spectrometer to yielddiagnostic patterns that can be matched to protein sequence databasesvia computer algorithms. The digested sample can be diluted to achieve 1ng or less starting material for a given protein. Biological samplescontaining a simple buffer background (e.g., water or volatile salts)are amenable to direct in-solution digest; more complex backgrounds(e.g., detergent, non-volatile salts, glycerol) require an additionalclean-up step to facilitate the sample analysis.

Patterns of protein fragments, or whole proteins, can be compared toknown controls for a given protein and identity can be determined bycomparison.

Example 12

Synthesis of mRNA Encoding Citrin

Sequence optimized polynucleotides encoding Citrin polypeptides, i.e.,SEQ ID

NOs: 1 and 3, are synthesized and characterized as described in Examples1 to 11. mRNA's encoding both human Citrin are prepared for Examples13-19 described below, and are synthesized and characterized asdescribed in Examples 1 to 11.

An mRNA encoding human Citrin is constructed, e.g., by using the ORFsequence provided in SEQ ID NO: 2 or 4. The mRNA sequence includes both5′ and 3′ UTR regions (see, e.g., SEQ ID NOs: 79 and 80, respectively).In a construct, the 5′UTR and 3′UTR sequences are:

5′UTR (SEQ ID NO: 79)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC 3′UTR (SEQ ID NO: 80)UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC  

The Citrin mRNA sequence is prepared as modified mRNA. Specifically,during in vitro translation, modified mRNA is generated using5-methoxy-UTP to ensure that the mRNAs contain 100% 5-methoxy-uridineinstead of uridine. Further, Citrin-mRNA is synthesized with a primerthat introduces a polyA-tail, and a Cap 1 structure is generated on bothmRNAs using Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferaseto generate: m7G(5′)ppp(5′)G-2′-O-methyl.

Example 13 Detecting Endogenous Citrin Expression In Vitro

Citrin expression is characterized in a variety of cell lines derivedfrom both mice and human sources. Cell are cultured in standardconditions and cell extracts are obtained by placing the cells in lysisbuffer. For comparison purposes, appropriate controls are also prepared.To analyze Citrin expression, lysate samples are prepared from thetested cells and mixed with lithium dodecyl sulfate sample loadingbuffer and subjected to standard Western blot analysis. For detection ofCitrin, the antibody used is a commercial anti-Citrin antibody. Fordetection of a load control, the antibody used is anti-citrase synthase(rabbit polyclonal; PA5-22126; Thermo-Fisher Scientific®). To examinethe localization of endogenous Citrin, immunofluorescence analysis isperformed on cells. Citrin expression is detected using a commercialanti-Citrin. The location of specific organelles can be detected withexisting commercial products. For example, mitochondria can be detectedusing Mitotracker, and the nucleus can be stained with DAPI. Imageanalysis is performed on a Zeiss ELYRA imaging system.

Endogenous Citrin expression can be used as a base line to determinechanges in Citrin expression resulting from transfection with mRNAscomprising nucleic acids encoding Citrin.

Example 14 In Vitro Expression of Citrin in HeLa Cells

To measure in vitro expression of human Citrin in HeLa cells, thosecells are seeded on 12-well plates (BD Biosciences, San Jose, USA) oneday prior to transfection. mRNA formulations comprising human Citrin ora GFP control are transfected using 800 ng mRNA and 2 μL Lipofectamin2000 in 60 μL OPTI-MEM per well and incubated.

After 24 hours, the cells in each well are lysed using a consistentamount of lysis buffer. Appropriate controls are used. Proteinconcentrations of each are determined using a BCA assay according tomanufacturer's instructions. To analyze Citrin expression, equal loadsof each lysate (24 μg) are prepared in a loading buffer and subjected tostandard Western blot analysis. For detection of Citrin, a commercialanti-Citrin antibody is used according to the manufacturer'sinstructions.

Example 15 In Vitro Citrin Activity in HeLa Cells

An in vitro Citrin activity assay is performed to determine whetherCitrin exogenously-expressed after introduction of mRNA comprising aCitrin sequence is active.

A. Expression Assay

HeLa cells are transfected with mRNA formulations comprising humanCitrin or a GFP control. Cells are transfected with Lipofectamin 2000and lysed as described in Example 14 above. Appropriate controls arealso prepared.

B. Activity Assay

To assess whether exogenous Citrin can function, an in vitro activityassay is performed using transfected HeLa cell lysates as the source ofenzymatic activity. To begin, lysate is mixed Citrin substrate. Thereaction is stopped by adding 100 g/L TCA and vortexing. The reactiontubes are then centrifuged at 13,000 g for 1 min, and the supernatant isanalyzed for the presence of labeled enzymatic products resulting fromthe activity of Citrin using HPLC-based separation and quantification.Specifically, 20 μL of each activity reaction supernatant are analyzedusing a HPLC system equipped with a Quaternary-Pump, a Multi-sampler, aThermostated Column-Compartment, a Poroshell EC-C18 120 HPLC-column anda Radiometric Detector controlled by OpenLAB Chromatography Data System,all used according to the manufacturers' recommendations.

Example 16 Measuring In Vitro Expression of Citrin in Cells

Cells from normal subjects and Citrullinemia patients are examined fortheir capacity to express exogenous Citrin. Cells are transfected withmRNA formulations comprising human Citrin, mouse Citrin, or a GFPcontrol via electroporation using a standard protocol. Each construct istested separately. After incubation, cells are lysed and proteinconcentration in each lysate is measured using a suitable assay, e.g.,by BCA assay. To analyze Citrin expression, equal loads of each lysateare prepared in a loading buffer and subjected to standard Western blotanalysis. For detection of Citrin, an anti-Citrin is used. For detectionof a load control, the antibody used is anti-citrase synthase (rabbitpolyclonal; MA5-17625; Pierce®).

Example 17 Measuring In Vitro Citrin Activity in Lysates A. Expression

Cells from normal human subjects and Citrullinemia patients arecultured. Cells are transfected with mRNA formulations comprising humanCitrin, mouse Citrin, or a GFP control via electroporation using astandard protocol.

B. Activity Assay

To assess whether exogenous Citrin function, an in vitro activity assayis performed using transfected cell lysates as the source of enzymaticactivity. Lysate containing expressed Citrin protein is incubated withlabeled Citrin substrate, and the activity of Citrin is quantified bymeasuring the levels of labeled products resulting from the enzymaticactivity of Citrin.

Example 18 In Vivo Citrin Expression in Animal Models

To assess the ability of Citrin-containing mRNA's to facilitate Citrinexpression in vivo, mRNA encoding human Citrin is introduced intoC57B/L6 mice. C57B/L6 mice are injected intravenously with eithercontrol mRNA (NT-FIX) or human Citrin mRNA. The mRNA is formulated inlipid nanoparticles for delivery into the mice. Mice are sacrificedafter 24 or 48 hrs. and Citrin protein levels in liver lysates aredetermined by capillary electrophoresis (CE). Citrate synthaseexpression is examined for use as a load control. For control NT-FIXinjections, 4 mice are tested for each time point. For human Citrin mRNAinjections, 6 mice are tested for each time point. Treatment with mRNAencoding Citrin is expected to reliably induce expression of Citrin.

Example 19 Human Citrin Mutant and Chimeric Constructs

A polynucleotide of the present invention can comprise at least a firstregion of linked nucleosides encoding human Citrin, which can beconstructed, expressed, and characterized according to the examplesabove. Similarly, the polynucleotide sequence can contain one or moremutations that results in the expression of a Citrin with increased ordecreased activity. Furthermore, the polynucleotide sequence encodingCitrin can be part of a construct encoding a chimeric fusion protein.

Example 20 Production of Nanoparticle Compositions A. Production ofNanoparticle Compositions

Nanoparticles can be made with mixing processes such as microfluidicsand T-junction mixing of two fluid streams, one of which contains thepolynucleotide and the other has the lipid components.

Lipid compositions are prepared by combining an ionizable amino lipiddisclosed herein, e.g., a lipid according to Formula (I) such asCompound 18, or a lipid according to Formula (III) such as Compound 236,a phospholipid (such as DOPE or DSPC, obtainable from Avanti PolarLipids, Alabaster, Ala.), a PEG lipid (such as1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known asPEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, Ala. orCompound 428), and a structural lipid (such as cholesterol, obtainablefrom Sigma-Aldrich, Taufkirchen, Germany, or a corticosteroid (such asprednisolone, dexamethasone, prednisone, and hydrocortisone), or acombination thereof) at concentrations of about 50 mM in ethanol.Solutions should be refrigerated for storage at, for example, −20° C.Lipids are combined to yield desired molar ratios and diluted with waterand ethanol to a final lipid concentration of between about 5.5 mM andabout 25 mM.

Nanoparticle compositions including a polynucleotide and a lipidcomposition are prepared by combining the lipid solution with a solutionincluding the a polynucleotide at lipid composition to polynucleotidewt:wt ratios between about 5:1 and about 50:1. The lipid solution israpidly injected using a NanoAssemblr microfluidic based system at flowrates between about 10 ml/min and about 18 ml/min into thepolynucleotide solution to produce a suspension with a water to ethanolratio between about 1:1 and about 4:1.

For nanoparticle compositions including an RNA, solutions of the RNA atconcentrations of 0.1 mg/ml in deionized water are diluted in 50 mMsodium citrate buffer at a pH between 3 and 4 to form a stock solution.

Nanoparticle compositions can be processed by dialysis to remove ethanoland achieve buffer exchange. Formulations are dialyzed twice againstphosphate buffered saline (PBS), pH 7.4, at volumes 200 times that ofthe primary product using Slide-A-Lyzer cassettes (Thermo FisherScientific Inc., Rockford, Ill.) with a molecular weight cutoff of 10kD. The first dialysis is carried out at room temperature for 3 hours.The formulations are then dialyzed overnight at 4° C. The resultingnanoparticle suspension is filtered through 0.2 μm sterile filters(Sarstedt, Nümbrecht, Germany) into glass vials and sealed with crimpclosures. Nanoparticle composition solutions of 0.01 mg/ml to 0.10 mg/mlare generally obtained.

The method described above induces nano-precipitation and particleformation. Alternative processes including, but not limited to,T-junction and direct injection, can be used to achieve the samenano-precipitation.

B. Characterization of nanoparticle compositions

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) can be used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the nanoparticle compositions in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy can be used to determine theconcentration of a polynucleotide (e.g., RNA) in nanoparticlecompositions. 100 μL of the diluted formulation in 1×PBS is added to 900μL of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, theabsorbance spectrum of the solution is recorded, for example, between230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter,Beckman Coulter, Inc., Brea, Calif.). The concentration ofpolynucleotide in the nanoparticle composition can be calculated basedon the extinction coefficient of the polynucleotide used in thecomposition and on the difference between the absorbance at a wavelengthof, for example, 260 nm and the baseline value at a wavelength of, forexample, 330 nm.

For nanoparticle compositions including an RNA, a QUANT-IT™ RIBOGREEN®RNA assay (Invitrogen Corporation Carlsbad, Calif.) can be used toevaluate the encapsulation of an RNA by the nanoparticle composition.The samples are diluted to a concentration of approximately 5 μg/mL in aTE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of thediluted samples are transferred to a polystyrene 96 well plate andeither 50 μL of TE buffer or 50 μL of a 2% Triton X-100 solution isadded to the wells. The plate is incubated at a temperature of 37° C.for 15 minutes. The RIBOGREEN® reagent is diluted 1:100 in TE buffer,and 100 μL of this solution is added to each well. The fluorescenceintensity can be measured using a fluorescence plate reader (WallacVictor 1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) at anexcitation wavelength of, for example, about 480 nm and an emissionwavelength of, for example, about 520 nm. The fluorescence values of thereagent blank are subtracted from that of each of the samples and thepercentage of free RNA is determined by dividing the fluorescenceintensity of the intact sample (without addition of Triton X-100) by thefluorescence value of the disrupted sample (caused by the addition ofTriton X-100).

Exemplary formulations of the nanoparticle compositions are presented inthe TABLE 6 below. The term “Compound” refers to an ionizable lipid suchas MC3, Compound 18, or Compound 236. “Phospholipid” can be DSPC orDOPE. “PEG-lipid” can be PEG-DMG or Compound 428.

TABLE 6 Exemplary Formulations of Nanoparticles Composition (mol %)Components 40:20:38.5:1.5 Compound:Phospholipid:Chol:PEG-Lipid45:15:38.5:1.5 Compound:Phospholipid:Chol:PEG-Lipid 50:10:38.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 55:5:38.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 60:5:33.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 45:20:33.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 50:20:28.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 55:20:23.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 60:20:18.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 40:15:43.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 50:15:33.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 55:15:28.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 60:15:23.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 40:10:48.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 45:10:43.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 55:10:33.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 60:10:28.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 40:5:53.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 45:5:48.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 50:5:43.5:1.5Compound:Phospholipid:Chol:PEG-Lipid 40:20:40:0Compound:Phospholipid:Chol:PEG-Lipid 45:20:35:0Compound:Phospholipid:Chol:PEG-Lipid 50:20:30:0Compound:Phospholipid:Chol:PEG-Lipid 55:20:25:0Compound:Phospholipid:Chol:PEG-Lipid 60:20:20:0Compound:Phospholipid:Chol:PEG-Lipid 40:15:45:0Compound:Phospholipid:Chol:PEG-Lipid 45:15:40:0Compound:Phospholipid:Chol:PEG-Lipid 50:15:35:0Compound:Phospholipid:Chol:PEG-Lipid 55:15:30:0Compound:Phospholipid:Chol:PEG-Lipid 60:15:25:0Compound:Phospholipid:Chol:PEG-Lipid 40:10:50:0Compound:Phospholipid:Chol:PEG-Lipid 45:10:45:0Compound:Phospholipid:Chol:PEG-Lipid 50:10:40:0Compound:Phospholipid:Chol:PEG-Lipid 55:10:35:0Compound:Phospholipid:Chol:PEG-Lipid 60:10:30:0Compound:Phospholipid:Chol:PEG-Lipid

Example 21 Evaluation of mRNA Encoding Citrin in a Citrin DeficiencyMouse Model

Modified mRNA constructs including sequence optimized ORFs encodinghuman Citrin (SLC25A13) isoform 1 are shown in TABLE 7. These constructswere used in Examples 21-24 as indicated. Each of constructs #1-#4comprises a Cap1 5′ terminal cap and a 3′ terminal poly-A region. Eachof constructs #1-#4 comprises a 5-methoxyuridine modified sequence.

TABLE 7 Modified mRNA constructs including optimized ORFs encoding humanCitrin. Citrin Citrin Full mRNA 5′UTR ORF 3′UTR construct construct SEQID NO SEQ ID NO SEQ ID NO SEQ ID NO #1 30 129 105 130 #2 30 132 105 133#3 30 135 105 136 #4 30 138 105 139

Blood ammonium level, aversion to sucrose (preference index), and bodyweight were assessed in double knockout mice lacking functional citrinand mitochondrial glycerol 3-phosphate dehydrogenase (mGPD) genes andcitrin knock-out mice after administration of Citrin mRNA or controlmRNA according to the study design shown in FIG. 7. Citrin knock-outmice do not display features of human citrin deficiency. However,mGPD/CTRN knockout mice used in this study display citrin deficiencyphenotypes, such as sucrose aversion and growth retardation, and severalaberrations in plasma biomarkers relative to wild-type mice, includingfor example, hypoinsulinemia under fed conditions; an increase in plasmatriglyceride; an increase in plasma free fatty acids (FFA), aspartateaminotransferase (AST), alanine aminotransferase (ALT) and glycerolunder fasted conditions; plasma hyperammonemia under fed conditions; anincrease in hepatic lactate and pyruvate; an increase in hepatictriglyceride levels at 16 hours post fasting; and an increase in hepaticcitrulline and glyceraldehyde 3-phosphate (G3P). Knock out mice weregenerated as described in Saheki et al., JBC 282(34):25041-25052 (2007);Saheki et al., Mol. Genetics and Metabolism 120:306-316 (2017) andMoriyama et al., Biochimica et Biophysica Acta 1852:1787-1795 (2015).

Mice injected (IV) with 0.5 mg/kg mRNA encoding human Citrin (Construct#1, formulated in lipid nanoparticle Compound 18) or control mRNA(NTFIX) were sacrificed according to the schedule provided in FIG. 7. Inshort, WT-NtFix (n=4), Ctrn KO-NtFix (n=2), Ctrn KO-hCitrin (n=2),Double KO-NtFix (n=5), and Double KO-hCitrin (n=5) mice were bled ondays −7, 2, 9, and 16, and administered 0.5 mg/kg mRNA on days 1, 8, and15. Blood ammonium (NH₄ ⁺) was measured on days 2, 9, and 16. Aversionto a 20% sucrose solution was assessed after the second injection ofmRNA according to the method described in Saheki et al., Mol. Geneticsand Metabolism 120:306-316 (2017). Mice were challenged with a 50%sucrose solution on day 16, and blood samples were collected one hourpost-challenge. In this terminal study, mice were sacrificed on day 16,body weight was determined, and plasma, liver, and spleen samples werecollected.

Blood ammonium levels are shown in FIG. 8. IV administration of mRNAencoding Citrin resulted in a 62% decrease in the blood ammonium levelin double knockout mice compared blood ammonium levels in doubleknockout mice administered control mRNA (NTFIX). Ammonium levels inWT-NtFix, Ctrn KO-NtFix, and Ctrn KO-hCitrin are shown for comparison.

The oral aversion of mGPD/CTRN knockout mice to sucrose was determinedbefore and after administration of mRNA via the method described inSaheki et al. Mol Genet Metab. 120(4):306-316 (2017). Briefly, mice wereprovided two drinking bottles following the second injection of mRNA(see study design in FIG. 7), one bottle containing water and the othercontaining a 50% sucrose solution. The preference index for sucrosesolution was calculated as (g of sucrose solution)/[(g of sucrosesolution)+(g of water)]. Wild-type mice and Citrin knockout miceexhibited an almost 100% preference for the sucrose solution (data notshown). mGPD/CTRN knockout mice administered NtFIX mRNA exhibited adecreased preference for the sucrose solution. Administration of mRNAencoding Citrin reversed the oral aversion of mGPD/CTRN knockout mice tosucrose (FIGS. 9A-9B).

Example 22 Determination of In Vivo Citrin Protein Expression in MiceAdministered mRNA Encoding Citrin

Citrin protein expression in isolated liver mitochondria was measured byWestern blot following mRNA administration according to the schedule inFIG. 7. Briefly, wild-type, CTRN knockout, and mGPD/CTRN knockout micewere administered control mRNA (NTFIX) or mRNA encoding Citrin(Construct #1, formulated in lipid nanoparticle Compound 18). Mice weresacrificed on day 16 and liver extracts were prepared. Citrin wasdetected by Western blot using an anti-Citrin antibody provided by Dr.Takeyori Saheki from the Kagoshima University Graduate School of Medicaland Dental Sciences (rabbit polyclonal antibody raised against theN-terminal half of human Citrin, as described in Yasuda et al., HumanGenetics 107:537-545 (2000)) (FIG. 10, top panel). mGPD proteinexpression was also monitored to ensure the complete deletion of thegene in mGPD/CTRN knockout mice (FIG. 10, middle panel). CitrateSynthase was detected in all samples as a control to normalize theprotein expression level of Citrin (FIG. 10, lower panel). Antibodiesfor detection of mGPD and Citrate were purchased from Novus Biologicals(NBP1-86121) and Thermo Fisher Scientific (MA5-17265), respectively.Administration of mRNA encoding human Citrin to CTRN knockout mice andmGPD/CTRN knockout mice resulted in increased Citrin protein expressioncompared to control mRNA NTFIX.

Semi-quantitative analysis of Citrin expression was performed on Westernblot signals from isolated liver michondria. Administration of mGPD/CTRNdouble knockout mice with mRNA encoding Citrin increased the relativeexpression of Citrin compared to control mRNA (NTFIX) (FIG. 11). Anincrease in Citrin expression was detected for mGPD/CTRN knockout miceadministered mRNA encoding Citrin.

Example 23 Multiple Dosing of mRNA Encoding Citrin in mGPD/CTRN DoubleKnockout Mice

The effect of multiple doses of mRNA encoding Citrin on plasma ammoniawas assessed in mGPD/CTRN knockout mice. Mice administered mRNA encodinghuman Citrin (Construct #1, formulated in lipid nanoparticle Compound18) or control mRNA (NTFIX) were injected and challenged according tothe timing schedule shown in FIG. 12. In short, WT mice were injectedwith 0.5 mg/kg NTFIX mRNA (n=4); mGPD/CTRN double KO mice were injectedwith 0.5 mg/kg NTFIX mRNA (n=5); mGPD/CTRN double KO mice were injectedwith 0.5 mg/kg Citrin mRNA (Construct #1) (n=5); and mGPD/CTRN double KOmice were injected with 0.2 mg/kg Citrin mRNA (Construct #1) (n=5).Following injections, mice were challenged with a solution containing50% sucrose. Blood plasma was obtained from the mice 24 hours after thefirst and final injections and plasma levels of ammonia were determined.

Prior to injection, there was no difference in plasma ammonia levels indifferent groups of mice (FIG. 13A). As expected, in response to thesucrose challenge, there was a significant increase in blood ammonialevels of the mGPD/CTRN double knockout mice treated with ntFIX (FIG.13A). However, as shown in FIG. 13A, plasma ammonia levels decreased ina dose-dependent manner following a single initial injection of CitrinmRNA, with a significant (P<0.05) reduction in plasma ammonia levelsobserved in double knockout mice receiving 0.5 mg/kg Citrin mRNAcompared to control mRNA (NTFIX). After the final injection, Citrin mRNAtreatment also led to a numerical decrease in plasma ammonia after thesucrose challenge (FIG. 13B).

Example 24 Determination of In Vivo Citrin Protein Expression andActivity

The in vivo Citrin protein expression and activity was evaluated inwild-type mice. Mice were injected (IV) with 0.5 mg/kg mRNA encodinghuman Citrin (Constructs #1, 2, 3 or 4) formulated in lipid nanoparticleCompound 18 or 0.5 mg/kg control mRNA (NTFIX). Liver mitochondria wereisolated 24 hours following injection, and Citrin protein expression wasdetected by Western blot using an anti-Citrin antibody from NovusBiologicals (NBP1-33380), as shown in FIG. 14A. Expression of α-tubulinwas detected in all samples as a control. A quantitative analysis of theWestern blot is shown in FIG. 14B. Citrin protein expression wasmeasured as a ratio of the Citrin band intensity from mice administeredmRNA encoding Citrin to the band intensity from mice administeredcontrol mRNA (NTFIX). Constructs #1 and #2 showed the highest proteinexpression levels.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections can set forth one or morebut not all exemplary embodiments of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

1.-158. (canceled)
 159. A pharmaceutical composition comprising a lipidnanoparticle, wherein the lipid nanoparticle comprises a compound havingthe formula (I)

or a salt or stereoisomer thereof, wherein R₁ is selected from the groupconsisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂and R₃ are independently selected from the group consisting of H, C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle; R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a carbocycle,heterocycle, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; each R₅ is independently selected from the group consisting ofC₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independently selected fromthe group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—,—S—S—, an aryl group, and a heteroaryl group; R₇ is selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ is selected fromthe group consisting of C₃₋₆ carbocycle and heterocycle; R₉ is selectedfrom the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR, —S(O)₂R,—S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle; each R isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R′ is independently selected from the groupconsisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″is independently selected from the group consisting of C₃₋₁₄ alkyl andC₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and 13; and provided that when R₄ is —(CH₂)_(n)Q,—(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1,2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl whenn is 1 or 2, wherein the lipid nanoparticle comprises an mRNA thatcomprises an open reading frame (ORF) encoding a human Citrinpolypeptide, wherein the composition is suitable for administration to ahuman subject in need of treatment for citrullinemia type 2 (CTLN2).160. The pharmaceutical composition of claim 159, wherein the lipidnanoparticle comprises the compound is of Formula (IA):

or a salt or stereoisomer thereof, wherein l is selected from 1, 2, 3,4, and 5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.
 161. Apharmaceutical composition comprising an mRNA comprising an open readingframe (ORF) encoding a human Citrin polypeptide, wherein the compositionwhen administered as a single intravenous dose to a subject in needthereof is sufficient to reduce blood and/or plasma levels of: (i)ammonia at least 2-fold, at least 3-fold, at least 5-fold, at least10-fold, at least 15-fold, at least 20-fold, or at least 50-foldcompared to a reference ammonia blood and/or plasma level in a subjecthaving citrullinemia type 2 (CTLN2), for at least 24 hours, at least 48hours, at least 72 hours, at least 96 hours, or at least 120 hourspost-administration; and/or (ii) triglycerides at least 1.5-fold, atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 10-fold, or at least 20-fold compared to a reference triglycerideblood and/or plasma level in a subject having CTLN2, for at least 24hours post-administration, at least 48 hours, at least 72 hours, atleast 96 hours, or at least 120 hours post-administration.
 162. Apharmaceutical composition comprising an mRNA comprising an open readingframe (ORF) encoding a human Citrin polypeptide, wherein the compositionwhen administered as a single intravenous dose to a subject in needthereof is sufficient to increase the level of hepatic Citrin activityat least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 10-fold, at least 20-fold, or at least 50-foldcompared to a reference hepatic Citrin activity level in a subjecthaving citrullinemia type 2 (CTLN2) for at least 24 hourspost-administration.
 163. A pharmaceutical composition comprising anmRNA comprising an open reading frame (ORF) encoding a human Citrinpolypeptide, wherein the composition when administered as a singleintravenous dose to a subject in need thereof is sufficient to reduceblood and/or plasma levels of ammonia at least at least 30%, at least40%, at least 50%, at least 60%, at least 70%, or at least 80% comparedto a reference ammonia blood and/or plasma level in a subject havingcitrullinemia type 2 (CTLN2) at least 24 hours, at least 48 hours, atleast 72 hours, at least 96 hours, or at least 120 hourspost-administration.
 164. A pharmaceutical composition comprising anmRNA comprising an open reading frame (ORF) encoding a human Citrinpolypeptide, wherein the composition when administered as a singleintravenous dose to a subject in need thereof is sufficient to: (i)increase hepatic Citrin activity level at or above a normalphysiological Citrin activity level for at least 24 hours, at least 48hours, at least 72 hours, at least 96 hours, or at least 120 hourspost-administration, and/or (ii) increase hepatic Citrin activity levelto 50% or more of the normal hepatic Citrin activity level at least 24hours, at least 48 hours, at least 72 hours, or at least 96 hourspost-administration.
 165. An mRNA comprising an open reading frame (ORF)encoding a human Citrin polypeptide, wherein the ORF has at least 85%sequence identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 5 to 29, 129, 132, 135, and
 138. 166. The mRNAof claim 165, wherein the human Citrin polypeptide comprises SEQ ID NO:1or SEQ ID NO:3.
 167. A pharmaceutical composition comprising the mRNA ofclaim 165 and a delivery agent.
 168. A lipid nanoparticle comprising themRNA of claim
 165. 169. A method of treating citrullinemia type 2(CTLN2) in a human subject in need thereof, comprising administering tothe human subject an effective amount of the pharmaceutical compositionof claim
 167. 170. A method of treating citrullinemia type 2 (CTLN2) ina human subject in need thereof, comprising administering to the humansubject an effective amount of the lipid nanoparticle of claim
 168. 171.A method of treating citrullinemia type 2 (CTLN2) in a human subject inneed thereof, comprising administering to the human subject an effectiveamount of the pharmaceutical composition of claim
 161. 172. A method ofreducing an ammonia and/or triglyceride blood plasma level in a humansubject having citrullinemia type 2 (CTLN2), comprising administering tothe human subject an effective amount of the pharmaceutical compositionof claim
 161. 173. The method of claim 172, wherein: (i) the ammoniablood and/or plasma level is reduced at least 2-fold, at least 3-fold,at least 5-fold, at least 10-fold, at least 15 fold, at least 20-fold orat least 50-fold compared to a reference ammonia blood and/or plasmalevel in a subject having CTLN2, for at least 24 hours, at least 48hours, at least 72 hours, at least 96 hours, or at least 120 hourspost-administration, and/or (ii) the triglyceride blood and/or plasmalevel is reduced at least 1.5-fold, 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold comparedto a reference triglyceride blood and/or plasma level in a subjecthaving CTLN2, for at least 24 hours, at least 48 hours, at least 72hours, at least 96 hours, or at least 120 hours post-administration.174. A method of increasing the level of Citrin activity in a humansubject having citrullinemia type 2 (CTLN2), comprising administering tothe human subject an effective amount of the pharmaceutical compositionof claim
 162. 175. The method of claim 174, wherein the level of Citrinactivity is increased at least 1.5-fold, at least 2-fold, at least5-fold, at least 10-fold, at least 20-fold, or at least 50-fold ascompared to a reference Citrin activity level in a subject having CTLN2for at least 24 hours, at least 48 hours, at least 72 hours, at least 96hours, or at least 120 hours post-administration.